Poly(vinyl ester) polymers for in vivo nucleic acid delivery

ABSTRACT

The present invention is directed membrane active poly(vinyl ester) polymers and compositions for targeted delivery of RNA interference (RNAi) polynucleotides to cells in vivo. RNAi polynucleotides are conjugated to the poly(vinyl ester) polymers and the polymers are reversibly modified to enable in vivo targeted delivery. Membrane activity of the poly(vinyl ester) provides for movement of the RNAi polynucleotides from outside the cell to inside the cell. Reversible modification provides physiological responsiveness.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/527,703 filed 26 Aug. 2011.

BACKGROUND OF THE INVENTION

The delivery of polynucleotides and other substantially cell membraneimpermeable compounds into a living cell is highly restricted by thecomplex membrane system of the cell. Drugs used in antisense, RNAi, andgene therapies are relatively large hydrophilic polymers and arefrequently highly negatively charged. Both of these physicalcharacteristics preclude their direct diffusion across the cellmembrane. For this reason, the major barrier to polynucleotide deliveryis the delivery of the polynucleotide across a cell membrane to the cellcytoplasm or nucleus.

One means that has been used to deliver small nucleic acid in vivo hasbeen to attach the nucleic acid to either a small targeting molecule ora lipid or sterol. While some delivery and activity has been observedwith these conjugates, the nucleic acid dose required with these methodshas been prohibitively large for practical application.

Numerous transfection reagents have been developed that achievereasonably efficient delivery of polynucleotides to cells in vitro.However, in vivo delivery of polynucleotides using these sametransfection reagents is complicated and rendered ineffective by in vivotoxicity, serum interactions, and poor targeting. Transfection reagentsthat work well in vitro, cationic polymers and lipids, typically formlarge electrostatic particles and destabilize cell membranes. Thepositive charge of in vitro transfection reagents facilitatesassociation with nucleic acid via charge-charge (electrostatic)interactions thus forming the nucleic acid/transfection reagent complex.Positive charge is also beneficial for nonspecific binding of thevehicle to the cell and for membrane fusion, destabilization, ordisruption. Destabilization of membranes facilitates delivery of thesubstantially cell membrane impermeable polynucleotide across a cellmembrane. While these properties facilitate nucleic acid transfer invitro, they cause toxicity and ineffective targeting in vivo. Cationiccharge results in interaction with serum components, which causesdestabilization of the polynucleotide-transfection reagent interactionand poor bioavailability and targeting. Membrane activity oftransfection reagents, which can be effective in vitro, often leads totoxicity in vivo.

For in vivo delivery, the vehicle (nucleic acid and associated deliveryagent) should be small, less than 100 nm in diameter, and preferablyless than 50 nm. Even smaller complexes, less than 20 nm or less than 10nm would be more useful yet. Delivery vehicles larger than 100 nm havevery little access to cells other than blood vessel cells in vivo.Complexes formed by electrostatic interactions tend to aggregate or fallapart when exposed to physiological salt concentrations or serumcomponents. Further, cationic charge on in vivo delivery vehicles leadsto adverse serum interactions and therefore poor bioavailability.Interestingly, high negative charge can also inhibit in vivo delivery byinterfering with interactions necessary for targeting. Thus, nearneutral vehicles are desired for in vivo distribution and targeting.Without careful regulation, membrane disruption or destabilizationactivities are toxic when used in vivo. Balancing vehicle toxicity withnucleic acid delivery is more easily attained in vitro than in vivo.

Rozema et al., in U.S. Patent Publication 20040162260 demonstrated ameans to reversibly regulate membrane disruptive activity of a membraneactive polyamine by reversible conversion of primary amines to pairs ofcarboxyl groups (β carboxyl and γ carboxyl of 2-propionic-3-methylmaleicanhydride). Rozema et al. (Bioconjugate Chem. 2003, 14, 51-57) reportedthat the β carboxyl did not exhibit a full apparent negative charge andby itself was not able to inhibit membrane activity. The addition of theγ carboxyl group was reported to be necessary for effective membraneactivity inhibition. However, because the vehicle was highly negativelycharged, with both the nucleic acid and the modified polymer having highnegative charge density, this system was not efficient for in vivodelivery.

By substituting neutral hydrophilic targeting (galactose) and stericstabilizing (PEG) groups for the γ carboxyl of2-propionic-3-methylmaleic anhydride, Rozema et al. were able to retainoverall water solubility and reversible inhibition of membrane activitywhile incorporating effective in vivo hepatocyte cell targeting (U.S.Patent Publication 20080152661).

We now describe new membrane active polymers and compositions made withthe described polymers for use in delivery of nucleic acids to cells invivo. These new polymers provide improved therapeutic potential overthose previously described.

SUMMARY OF THE INVENTION

In a preferred embodiment, the invention features amphipathic cationicpoly(vinyl ester) random copolymers particularly suited for deliveringpolynucleotides to cells in vivo. An amphipathic cationic poly(vinylester) random copolymer of the invention comprises a plurality ofamine-containing vinyl ester monomers and a plurality of firsthydrophobic vinyl ester monomers. The amine-containing monomers containpendant primary amine groups. The hydrophobic monomers contain pendenthydrophobic groups having 2-20 carbon atoms selected from the groupconsisting of: hydrocarbon group, alkyl group, alkenyl group, alkynylgroup, alkoxy alkyl group, aromatic group, and aryl group. The polymersmay further comprise a plurality of second amine-containing vinyl estermonomers or a plurality of second hydrophobic vinyl ester monomers.Second amine-containing vinyl ester monomers contain pendant aminegroups selected from the group consisting of: primary amine, secondaryamine, tertiary amine, quaternary amine, protected amine, nitrogenheterocycle, aldimine, hydrazide, hydrazone, and imidazole. In additionto being amphipathic, the poly(vinyl ester) random copolymers of theinvention are membrane active. A preferred poly(vinyl ester) randomcopolymer comprises primary amine containing and butyryl vinyl estermonomers.

Poly(vinyl ester) random copolymers of the invention may be synthesizedfrom two, three, or four different monomers. Monomers may be selectedfrom the list comprising: protected amine vinyl ester, imidazole vinylester, alkyl vinyl ester, alkenyl vinyl ester, alkynyl vinyl ester,aromatic vinyl ester, and aryl vinyl ester. Protected amine vinyl estermonomers include, but are not limited to: tert-Butoxycabonyl (Boc)protected amine containing vinyl ester. Protected primary amine monomersare copolymerized with alkyl vinyl ester monomers. The amine protectinggroups are then removed post-polymerization to form aqueous soluble,amphipathic random copolymers. The aliphatic hydrophobic groups may belinear, branched, or cyclic and may contain one or more substitutions ofheteroatoms.

In a preferred embodiment, poly(vinyl ester) random copolymers aresynthesized by Reversible Addition-Fragmentation chain Transfer (RAFT)polymerization. In one embodiment, the RAFT polymerization is carriedout using Malonate N,N-diphenyl dithiocarbamate (MDP-DTC). Using RAFTpolymerization, and optionally fractionization, polymers having apolydispersity of less than 1.5, or more preferably less than 1.4 or1.3, are possible.

For delivery of a polynucleotide to a cell in vivo, the describedamphipathic poly(vinyl ester) random copolymers are reversibly modified.Reversible modification comprises attachment of a plurality of maskingagents, as defined herein, to polymer primary amines through a pluralityof reversible physiologically labile covalent bonds. Reversiblephysiologically labile covalent bonds may be selected from the groupcomprising: pH labile bonds and enzymatically cleavable bonds. As usedherein, reversible modification means polymer primary amines arerestored upon cleavage of the physiologically labile covalent bondlinking the masking agent to the polymer. In a preferred embodiment,more than 50%, more than 60%, more than 70%, more than 80%, or more than90% of polymer primary amines are modified by reversible attachment ofmasking agents. Masking agents may be selected from the groupcomprising: steric stabilizers and targeting groups. The masking agentsimprove biodistribution or targeting of the polymer or apolymer-polynucleotide conjugate in vivo. Masking agents may inhibitnon-specific interactions of the polymer with serum components ornon-target cells. Masking agents may reduce aggregation of the polymeror polymer-polynucleotide conjugate. Masking agents containing targetinggroups enhance cell-specific targeting or cell internalization bytargeting the conjugate system to a cell surface receptor. The maskingagents can be conjugated to the polymer prior to or subsequent toconjugation of the polymer to a polynucleotide.

In another preferred embodiment, a polynucleotide is linked to thepolymer of the invention through a second physiologically labilecovalent bond. One or more polynucleotides may be linked to the polymervia the second physiologically labile covalent bonds. The labile bondlinking the masking agent to the polymer, first labile bond, and thelabile bond linking the polynucleotide to the polymer, second labilebond, maybe cleaved under the same or similar conditions or they may becleaved under distinct conditions, i.e. they may be orthogonal labilebonds. The polynucleotide may be selected from the group comprising:DNA, RNA, blocking polynucleotide, oligonucleotide, RNA interferencepolynucleotide, siRNA, microRNA, mRNA, and shRNA. Second physiologicallylabile covalent bonds may be selected from the group comprising: pHlabile bonds, enzymatically cleavable bonds, disulfide bonds, andnucleic acid ester bonds.

In a preferred embodiment, we describe a composition comprising: anamphipathic poly(vinyl ester) random copolymer covalently linked to: a)one or more targeting groups and or steric stabilizers via reversiblephysiologically labile covalent bonds; and, b) one or morepolynucleotides via orthogonal second physiologically labile covalentbonds. The polynucleotide-polymer conjugate is administered to a mammalin a pharmaceutically acceptable carrier or diluent.

In a preferred embodiment, we describe a polymer conjugate system fordelivering a membrane impermeable molecule to a cell and releasing themolecule in the cell. The polymer conjugate system comprises themembrane impermeable molecule reversibly linked to a reversibly modifiedpoly(vinyl ester) of the invention. A preferred membrane impermeablemolecule comprises a polynucleotide. A preferred polynucleotidecomprises an RNA interference polynucleotide. A preferred RNAinterference polynucleotide comprises an siRNA or miRNA. The polymer orpolynucleotide-polymer conjugate is administered to a mammal in apharmaceutically acceptable carrier or diluent.

In another preferred embodiment, the invention features a compositionfor delivering an RNA interference polynucleotide to a liver cell invivo comprising: an amphipathic poly(vinyl ester) random copolymercovalently linked to: one or more targeting groups and/or stericstabilizers via reversible physiologically labile covalent bonds and anRNA interference polynucleotide conjugated to a polynucleotide targetinggroup (polynucleotide conjugate). A preferred polynucleotide targetinggroup is a hydrophobic group containing at least 20 carbon atoms.Another preferred polynucleotide targeting group is a trivalentgalactosamine. The poly(vinyl ester) and the polynucleotide-conjugateare synthesized separately and may be supplied in separate containers ora single container. In this composition, the polynucleotide is notconjugated to the polymer. The modified polymer andpolynucleotide-conjugate are administered to a mammal inpharmaceutically acceptable carriers or diluents. In one embodiment, thedelivery polymer and the RNAi polynucleotide conjugate may be combinedin a solution prior to administration to the mammal. In anotherembodiment, the delivery polymer and the RNAi polynucleotide conjugatemay be co-administered to the mammal in separate solutions. In yetanother embodiment, the delivery polymer and the RNAi polynucleotideconjugate may be administered to the mammal sequentially. For sequentialadministration, the delivery polymer may be administered prior toadministration of the RNAi polynucleotide conjugate. Alternatively, forsequential administration, the RNAi polynucleotide conjugate may beadministered prior to administration of the delivery polymer.

In another embodiment, the described amphipathic poly(vinyl ester)random copolymers are suitable for delivering polynucleotides tomammalian cells in vitro. For in vitro cell delivery, the amphipathicpoly(vinyl ester) random copolymers may be reversibly modified asdescribed or used without reversible modification. They may also becombined with lipids or other polymers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustration shown the structure of an various amphipathicpoly(vinyl ester) random copolymer wherein:

-   -   N is a primary amine having the form —NH₂,    -   N′ is a secondary, tertiary, or quaternary amine having the form        —NR⁵H, —NR⁵R⁶, or —NR⁵R⁶R⁷ (wherein R⁵, R⁶, and R⁷ are        independently selected from —CH₃ and —CH₂—CH₃,) or alternatively        N′ can be a nitrogen heterocycle, aldimine, hydrazide,        hydrazone, or imidazole,    -   Y and Y′ are linker groups,    -   R and R′ are hydrophobic groups independently having 2-20 carbon        atoms,    -   R1, R2, R3, and R4 are independently selected from hydrogen (—H)        and methyl (—CH₃),    -   m and p are integers greater than zero (0),    -   n and q are integers greater than or equal to zero (0), and    -   the ratio (m+n)/(p+q) is 1-9.

FIG. 2. Illustration showing the structures of various dipeptide maskingagents.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to amphipathic poly(vinyl ester) randomcopolymers and conjugate systems thereof useful for the delivery ofbiologically active substances, such as nucleic acids, peptides, andproteins. The delivery of nucleic acids and other substantially cellmembrane impermeable compounds into a living cell is highly restrictedby the complex membrane system of the cell. For in vivo delivery theamphipathic poly(vinyl ester) random copolymers are reversibly modifiedby covalent attachment of masking agents via physiologically labilelinkages.

In one embodiment, the present invention is directed to membrane activepoly(vinyl ester) random copolymers of formula (I):

wherein:

-   N is a primary amine having the form —NH₂,-   N′ is a secondary, tertiary, or quaternary amine having the form    —NR⁵H, —NR⁵R⁶, or —NR⁵R⁶R⁷ wherein R⁵, R⁶, and R⁷ are independently    selected from —CH₃ and —CH₂—CH₃, or alternatively N′ can be a    nitrogen heterocycle, aldimine, hydrazide, hydrazone, or imidazole,-   Y and Y′ are linker groups,-   R and R′ are hydrophobic groups as defined herein independently    having 2-20 carbon atoms, or alkoxyl ethyl groups,    —(CH₂)_(l)—O'CH₂—CH₃, wherein l is 2, 3, or 4 (a preferred alkoxy    ethyl group is a 2-ethoxyethyl group, —(CH₂)₂—O—CH₂—CH₃,-   R1, R2, R3, and R4 are independently selected from hydrogen (—H) and    methyl (—CH₃),

m and p are integers greater than zero (0),

n and q are integers greater than or equal to zero (0), and

the ratio (m+n)/(p+q) is 1-9 (50-90% amines) and more preferably 1.5-4(60-80% amines).

A preferred R group is a hydrophobic group having 2-6 carbon atoms.

Linker groups Y and Y′ are uncharged and link the nitrogen to the vinylester via 1-24 carbon atoms, one or more of which may be substituted forheteroatoms. In a preferred embodiment, Y and Y′ independently contain1-12 carbon atoms, one or more of which may be substituted forheteroatoms. In one embodiment, Y and Y′ are independently selected from—(CH₂)_(x)— and —(CH₂—CH₂—O)_(z)—(CH₂)_(x)—, wherein x and z areindependently 1, 2, 3, 4, 5, or 6.

In one embodiment, the present invention is directed to vinyl esterrandom copolymers of formula (Ia):

wherein:

-   -   N is a primary amine having the form —NH₂,    -   Y is a linker group as described above,    -   R is a hydrophobic group as defined herein having 2-6 carbon        atoms or an alkoxyl ethyl group, —(CH₂)_(l)—O—CH₂—CH₃, wherein l        is 2, 3, or 4 (preferably 2-ethoxyethyl),    -   R1 and R3 are independently selected from hydrogen (—H) and        methyl (—CH₃),    -   m is an integer greater than zero (0),    -   p is an integer greater than zero (0),    -   the ratio m/p is 1-9 (50-90% amines) and more preferably 1.5-4        (60-80% amines).

The polymers according to the present invention can be generallyobtained as described herein and using methods known to the person ofordinary skill in the art of organic or medicinal chemistry. Thepolymers are polymerized from hydrophobic group-containing vinyl estermonomers and protected amine-containing vinyl ester monomers.Polymerization to form the polymers of the invention is preferablycarried out using Reversible Addition-Fragmentation chain Transfer(RAFT) polymerization. In one embodiment, the RAFT polymerization iscarried out using Malonate N,N-diphenyl dithiocarbamate (MDP-DTC).Polymer synthesis is performed using protected amine monomers.Deprotection of the amine yields the amine-containing polymers offormulae (I) or (Ia), wherein N is —NH₂. As an example, compounds offormula (Ia), wherein Y is —(CH₂)₃—, R1 is hydrogen, R3 is hydrogen, andR is —(CH₂)₃—CH₃ and can be obtained using compounds 3 and 8 as startingmaterial.

Synthesis of vinyl ester monomers 3 and 8 are described below.

Reversible Addition-Fragmentation chain Transfer (RAFT) polymerizationis a form of controlled radical polymerization. More specifically, RAFTis a type of living polymerization involving a conventional radicalpolymerization in the presence of a reversible chain transfer reagent.RAFT polymerization permits synthesis of a wide range of polymers withcontrolled molecular weight and low polydispersity (PDI), between 1.05and 1.6, for many monomers. Poly(vinyl ester)s of the inventionpreferably have a polydispersity less than 1.5 and more preferably lessthan 1.4 or 1.3. Fractionation may be used to further reducepolydispersity. RAFT polymerization is described in WO9504026,WO9801478, WO9905099, WO9931144, WO10083569, U.S. Pat. Nos. 6,291,620,6,376,626, 6,642,318, and 6,747,111. Polymers with molecular weightsgreater than 20,000 and low polydisperity are also possible with RAFTpolymerization and are preferred for in vivo delivery. In order formacromolecules to circulate through the blood stream effectively and tonot be cleared by the kidneys, molecular weights above 30,000-50,000 areoften preferred.

It is an essential feature of the unmodified amphipathic poly(vinylester) random copolymers of the invention that they are membrane active;i.e., they are capable of disrupting plasma membranes orlysosomal/endocytic membranes. Membrane activity, however, can lead totoxicity when the polymer is administered in vivo. Polyamines alsointeract readily with many anionic components in vivo, leading toundesired bio-distribution. Therefore, reversible inhibition of membraneactivity of the polyamine is used for in vivo use. This inhibition isaccomplished through reversible physiologically labile attachment ofmasking agents to polymer amines to form a reversibly masked membraneactive poly(vinyl ester), i.e. herein also termed a delivery polymer. Inaddition to inhibiting membrane activity, the masking agents shield thepolymer from non-specific interactions, reduce serum interactions,increase circulation time, or provide cell-specific interactions, i.e.targeting. The process of reversible modification also reduces positivecharge to form a near neutral charge polymer. As used herein, labilemeans that linkage of the masking agent to the polymer is readilycleaved under conditions typically present under physiologicalconditions. As used herein, reversible means that cleavage of the bondlinking the masking agent to the polymer results in restoration of thepolymer amine to the pre-modified state, i.e. to a primary amine.

A preferred reversible physiologically labile linkage comprises: aphysiologically labile covalent bond or a covalent bond cleavable undermammalian intracellular conditions. A preferred labile covalent bondcomprises a pH labile bond. A preferred pH labile physiologically labilelinkage comprises a maleamate. Another preferred physiologically labilelinkage comprises an enzymatically cleavable linkage. A preferredenzymatically cleavable linkage is a peptide (amide) bond. A preferredpeptide linkage comprises a dipeptide-amidobenzyl-carbonate as describedin U.S. patent application Ser. No. 13/326,433, incorporated herein byreference.

It is an essential feature of the masking agents that, in aggregate,they inhibit membrane activity of the polymer, shield the polymer fromnon-specific interactions (reduce serum interactions, increasecirculation time), and provide in vivo cell targeting. The membraneactive poly(vinyl ester)s of the invention are membrane active in theunmodified (unmasked) state and not membrane active (inactivated) in themodified (masked) state. A sufficient number of masking agents arelinked to the polymer to achieve the desired level of inactivation. Thedesired level of modification of a poly(vinyl ester) by attachment ofmasking agent(s) is readily determined using appropriate membraneactivity assays. For example, if the poly(vinyl ester) possessesmembrane activity in a given assay, a sufficient level of masking agentis linked to the polymer to achieve the desired level of inhibition ofmembrane activity in that assay. Masking requires modification of ≧50%,≧60%, ≧70%, or ≧80% of the amine groups on the polymer, as determined bythe quantity of amines on the polymer in the absence of any maskingagents. It is also a preferred characteristic of masking agents thattheir attachment to the polymer reduces net charge of the polymer, thusforming a more neutral delivery polymer. It is desirable that the maskedpolymer retain aqueous solubility.

As used herein, a membrane active poly(vinyl ester) of the invention ismasked if the modified polymer does not exhibit membrane activity andexhibits cell-specific (e.g., hepatocyte) targeting in vivo. A membraneactive poly(vinyl ester) of the invention is reversibly masked ifcleavage of linkages attaching the masking agents to the polymer resultsin restoration of amines on the poly(vinyl ester) thereby restoringmembrane activity.

It is another essential feature that the masking agents are linked tothe membrane active poly(vinyl ester) through reversible physiologicallylabile covalent bonds. By using reversible physiologically labilelinkages or bonds, the masking agents can be cleaved from the polymer invivo, thereby unmasking the polymer and restoring activity of theunmasked polymer. By choosing an appropriate reversible linkage, it ispossible to form a conjugate that restores activity of the membraneactive polymer after it has been delivered or targeted to a desired celltype or cellular location. Reversibility of the linkages provides forselective activation of the membrane active polymer. Suitable reversiblecovalent linkages contain reversible labile bonds which may be selectedfrom the group comprising: physiologically labile bonds, cellularphysiologically labile bonds, protease sensitive linkages, pH labilebonds, very pH labile bonds, and extremely pH labile bonds.

As used herein, a masking agent comprises a compound having an celltargeting group or a steric stabilizer and an amine-reactive groupwherein reaction of the amine-reactive group with an amine on apoly(vinyl ester) results in linkage of the targeting group or stericstabilizer to the polymer via a reversible physiologically labilecovalent bond. Preferably, the masking agent is charge neutral. Apreferred targeting group is an Asialoglycoprotein Receptor (ASGPr)targeting group. An ASGPr targeting group is a group, typically asaccharide, having affinity for the asialoglycoprotein receptor. Apreferred steric stabilizer is a polyethylene glycol (PEG). Preferredmasking agents of the invention are able to modify the poly(vinylester)s of the invention (form a reversible bond with the polymer) inaqueous solution.

A preferred amine-reactive group comprises a disubstituted maleicanhydride. A preferred masking agent is represented by the structure:

wherein R¹ is an alkyl group such as a methyl group (—CH₃), ethyl group(—CH₂CH₃), or propyl group (—CH₂CH₂CH₃), and R² comprises a neutraltargeting group or a neutral steric stabilizer. More preferably, thetargeting agent and steric stabilizer are uncharged. Monosubstitutedmaleic anhydrides, in which R1 or R2 is a hydrogen, yield linkages whichare not suitable for the described invention. While reaction of a maleicanhydride with an amine yields a β carboxyl group, this β carboxyl doesnot exhibit a full apparent negative charge (Rozema et al. BioconjugateChem. 2003, 14, 51-57). Therefore, maleic anhydride-based masking agentsin which R1 and R2 are charge neutral can be used to neutralize apolyamine without imparting high negative charge.

In one embodiment, poly(vinyl ester) polyamines of the invention arereversibly modified by reaction with a plurality of disubstituted maleicanhydrides. The present invention therefore provides random copolymersof formulae:

wherein N′, Y, Y′, R, R′, R1, R2, R3, R4, m, n, p, q have the meaningsgiven for formulae (I) and (Ia) above,

-   -   m1 is an integer≧zero and ≦m of formula (I) or (Ia),    -   m3 is an integer≧zero and ≦m of formula (I) or (Ia),    -   m1+m2+m3=m of formulae (I) or (Ia),    -   m1+m3 is an integer≧m2[i.e., ≧0.5×m of formulae (I) or (Ia) and        ≦m of formula (I) or (Ia)],    -   R7 is an alkyl group and R8 comprises a neutral targeting group        or R8 is an alkyl group and R7 comprises a neutral targeting        group, and    -   R9 is an alkyl group and R10 comprises a neutral steric        stabilizer or R10 is an alkyl group and R9 comprises a neutral        steric stabilizer.

Another preferred masking agent comprises a protease sensitivedipeptide-amidobenzyl-carbonate represented by the structure:

wherein R4 comprises a neutral, preferably uncharged, targeting ligandor steric stabilizer, R3 comprises an amine reactive carbonate group,and R1 and R2 are amino acid side chains. In a preferred dipeptide, R1is a hydrophobic amino acid side chain and R2 is an unchargedhydrophilic amino acid side chain. A preferred hydrophobic amino acid isphenylalanine, valine, isoleucine, leucine, alanine, or tryptophan. Apreferred uncharged hydrophilic amino acid is asparagine, glutamine, orcitrulline. A more preferred hydrophobic amino acid is phenylalanine orvaline. A more preferred uncharged hydrophilic amino acid is citrulline.A preferred activated carbonate is a para-nitrophenol. However, otheramine reactive carbonates known in the art are readily substituted forthe para-nitrophenol. Reaction of the activated carbonate with an amineconnects the targeting ligand or steric stabilizer to the membraneactive polyamine via a peptidase cleavable dipeptide-amidobenzylcarbamate linkage. Enzyme cleavage of the dipeptide, between the aminoacid and the amidobenzyl group removes R4 from the polymer and triggersan elimination reaction which results in regeneration of the polymeramine.

Reaction of a dipeptide-amidobenzyl-carbonate masking agent with anamine of the poly(vinyl ester) results in reversible modification of thepoly(vinyl ester). Hence, provided herein are conjugates comprising theamphipathic membrane active poly(vinyl ester)s described herein maskedby modification with dipeptide-amidobenzyl-carbonate masking agents. Thepolymers so masked have the formula:

wherein:

-   -   X is —NH—, —O—, or —CH₂—    -   Y is —NH— or —O—    -   R1 is preferably        -   —(CH₂)_(k)— phenyl (k is 1, 2, 3, 4, 5, 6; k=1            phenylalanine),        -   —CH—(CH₃)₂ (valine),        -   —CH₂—CH—(CH₃)₂ (leucine),        -   —CH(CH₃)—CH₂—CH₃ (isoleucine),        -   —CH₃ (alanine),        -   —(CH₂)₂—COOH (glutamic acid),        -   or

-   -   R2 is preferably        -   hydrogen (glycine)        -   —(CH₂)₃—NH—C(O)—NH₂ (citrulline),        -   —(CH₂)₄—N—(CH₃)₂ (lysine(CH₃)₂),        -   —(CH2)_(k)—C(O)—NH₂; (k is 1, 2, 3, 4, 5, 6),        -   —CH₂—C(O)—NH₂ (asparagine),        -   —(CH₂)₂—C(O)—NH₂ (glutamine),        -   —CH₂—C(O)—NR¹R² (aspartic acid amide),        -   —(CH₂)₂—C(O)—NR¹R² (glutamic acid amide),        -   —CH₂—C(O)—OR¹ (aspartic acid ester), or        -   —(CH₂)₂—C(O)—OR' (glutamic acid ester),            -   R¹ and R² are alkyl groups    -   R4 comprises a neutral polyethylene glycol or targeting ligand;        and the polyamine is an amphipathic membrane active poly(vinyl        ester).

While the structure above indicates a single dipeptide masking agentlinked to the polymer, in practice of the invention, 50% to 90% or moreof polymer amines are modified by dipeptide masking agents.

The membrane active poly(vinyl ester)s of the invention can beconjugated to masking agents in the presence of an excess of maskingagents. The excess masking agent may be removed from the conjugateddelivery polymer prior to administration of the delivery polymer.

In one embodiment, the membrane active poly(vinyl ester) polyamine isreversibly masked by attachment of targeting group masking agents orsteric stabilizer masking agents to ≧50%, ≧60%, ≧70%, or ≧80% of amineson the polyamine. In another embodiment, the membrane active polyamineis reversibly masked by attachment of a combination of targeting groupmasking agents and steric stabilizer masking agents to ≧50%, ≧60%, ≧70%,or ≧80% of amines on the polyamine. In another embodiment, the targetinggroup masking agents comprise a targeting group linked to anamine-reactive group via a PEG linker. For membrane active polyaminemasking with both targeting group masking agents and steric stabilizermasking agents, a ratio of steric stabilizer to targeting group is about0-4:1, more preferably about 0.5-2:1. In another embodiment, there areabout 1.3-2 steric stabilizer masking agents to about 1 targeting groupagent.

In a further embodiment of the present invention, there is provided aconjugate of the polymers of formula (I) or (Ia) covalently attached toa biologically active compound, preferably an RNA interferencepolynucleotide. Preferably, the polymer is covalently linked to thepolynucleotide by a physiologically labile linkage. A preferredphysiologically labile linkage is orthogonal to the masking agentphysiologically labile linkage. A suitable physiologically labilelinkage may be selected from the group comprising: physiologicallylabile bonds, cellular physiologically labile bonds, pH labile bonds,very pH labile bonds, extremely pH labile bonds, enzymatically cleavablebonds (including appropriate ester, amide, and phopshodiester bonds),and disulfide bonds.

We have found that by attaching the polynucleotide to the polymer via areversible linker that is broken after the polynucleotide is deliveredto the cell, it is possible to deliver a functionally activepolynucleotide to a cell in vivo. The labile linker is selected suchthat it undergoes a chemical transformation (e.g., cleavage) whenpresent in certain physiological conditions, (e.g., the reducingenvironment of the cell cytoplasm). Attachment of a polynucleotide topoly(vinyl ester) of the invention enhances delivery of thepolynucleotide to a cell in vivo. Release of the polynucleotide from thepolymer, by cleavage of the labile linkage, facilitates interaction ofthe polynucleotide with the appropriate cellular components foractivity.

The RNAi polynucleotide-polymer conjugate is formed by linking the RNAipolynucleotide to the polymer via a physiologically labile covalentbond. The polynucleotide is synthesized or modified such that itcontains a reactive group A. The polymer is also synthesized or modifiedsuch that it contains a reactive group B. Reactive groups A and B arechosen such that they can be linked via a physiologically labilecovalent linkage using methods known in the art. The polymer may belinked to the 3′ or the 5′ end of the RNAi polynucleotide. For siRNApolynucleotides, the targeting group may be linked to either the sensestrand or the antisense strand, though the sense strand is preferred.

Conjugation of the RNAi polynucleotide to a side chain primary amine ofpolymers (I) or (Ia) results in polymers of formula (IV) or (IVa).

-   -   wherein N′, Y, Y′, R, R′, R1, R2, R3, R4, m, n, p, q have the        meanings given for formulae (I) and (Ia) above,    -   m1 is 1, 2, 3, or 4,    -   m1+m2=m of formula (I) or (Ia); and    -   the linker comprises a physiologically labile linker.

In another embodiment, the RNAi polynucleotide is conjugated to apolymer backbone terminus as illustrated in formulae (V) and (Va). Thepolynucleotide may also be attached to the other terminus.

wherein N, N′, Y, Y′, R, R′, R1, R2, R3, R4, m, n, p, q have themeanings given for formulae (I) and (Ia) above, and the linker comprisesa physiologically labile linker.

In a further embodiment of the present invention, there are providedconjugates of the polymers of formulae (II), (IIa), and (III) covalentlyattached to a biologically active compound, preferably an RNAinterference polynucleotide, as shown above for the unmodified polymers.Preferably, the polymer is covalently linked to the polynucleotide by aphysiologically labile linkage.

The polynucleotide can be attached to the polymer in the presence of anexcess of polymer. The excess polymer may aid in formulation of thepolynucleotide-polymer conjugate. The excess polymer may reduceaggregation of the conjugate during formulation of the conjugate. Thepolynucleotide-polymer conjugate may be separated from the excesspolymer prior to administration of the conjugate to the cell ororganism. Alternatively, the polynucleotide-polymer conjugate may beco-administered with the excess polymer to the cell or organism. Theexcess polymer may be the same as the polymer or it may be different, ahelper or boost polymer.

In another embodiment, the invention features compositions fordelivering RNA interference polynucleotides to a liver cells in vivocomprising: a polymer of formula (II), (IIa), or (III), and an RNAinterference polynucleotide conjugated to a polynucleotide targetinggroup. The polynucleotide targeting group can be either a hydrophobicgroup containing at least 20 carbon atoms or a trivalent ASPGr targetinggroup as described in U.S. Patent Publication 20110207799. Thereversibly modified poly(vinyl ester) and the siRNA-conjugate aresynthesized separately and may be supplied in separate containers or asingle container. The RNA interference polynucleotide is not conjugatedto the polymer.

We have found that conjugation of an RNAi polynucleotide to apolynucleotide targeting group, either a hydrophobic group or to agalactose cluster, and co-administration of the RNAi polynucleotideconjugate with the modified poly(vinyl ester) polymers described aboveprovides for efficient, functional delivery of the RNAi polynucleotideto liver cells, particularly hepatocytes, in vivo. By functionaldelivery, it is meant that the RNAi polynucleotide is delivered to thecell and has the expected biological activity, sequence-specificinhibition of gene expression. Many molecules, includingpolynucleotides, administered to the vasculature of a mammal arenormally cleared from the body by the liver. Clearance of apolynucleotide by the liver wherein the polynucleotide is degraded orotherwise processed for removal from the body and wherein thepolynucleotide does not cause sequence-specific inhibition of geneexpression is not considered functional delivery.

The RNAi polynucleotide-polynucleotide targeting group conjugate isco-administered with a reversibly modified poly(vinyl ester) of theinvention. By co-administered it is meant that the RNAi polynucleotideand the delivery polymer are administered to the mammal such that bothare present in the mammal at the same time. The RNAipolynucleotide-targeting group conjugate and the delivery polymer may beadministered simultaneously or they may be delivered sequentially. Forsimultaneous administration, they may be mixed prior to administration.For sequential administration, either the RNAi polynucleotide-targetinggroup conjugate or the delivery polymer may be administered first.

For RNAi polynucleotide-hydrophobic targeting group conjugates, the RNAiconjugate may be administered up to 30 minutes prior to administrationof the delivery polymer. Also for RNAi polynucleotide-hydrophobictargeting group conjugates, the delivery polymer may be administered upto two hours prior to administration of the RNAi conjugate.

For RNAi polynucleotide-galactose cluster targeting group conjugates,the RNAi conjugate may be administered up to 15 minutes prior toadministration of the delivery polymer. Also for RNAipolynucleotide-galactose cluster targeting group conjugates, thedelivery polymer may be administered up to 15 minutes prior toadministration of the RNAi conjugate.

Amphipathic

The poly(vinyl ester) random copolymers of the invention areamphipathic. Amphipathic, or amphiphilic, polymers and have bothhydrophilic (polar, water-soluble) and hydrophobic (non-polar,lipophilic, water-insoluble) groups or parts.

As used herein, with respect to amphipathic polymers, a part is definedas a molecule derived when one covalent bond is broken and replaced byhydrogen. For example, in butyl amine, a breakage between the carbon andnitrogen bonds, and replacement with hydrogens, results in ammonia(hydrophilic) and butane (hydrophobic). If 1,4-diaminobutane is cleavedat nitrogen-carbon bonds, and replaced with hydrogens, the resultingmolecules are again ammonia (2×) and butane. However, 1,4,-diaminobutaneis not considered amphipathic because formation of the hydrophobic partrequires breakage of two bonds.

Membrane Active

As used herein, membrane active polymers are surface active, amphipathicpolymers that are able to induce one or more of the following effectsupon a biological membrane: an alteration or disruption of the membranethat allows non-membrane permeable molecules to enter a cell or crossthe membrane, pore formation in the membrane, fission of membranes, ordisruption or dissolving of the membrane. As used herein, a membrane, orcell membrane, comprises a lipid bilayer. The alteration or disruptionof the membrane can be functionally defined by the polymer's activity inat least one the following assays: red blood cell lysis (hemolysis),liposome leakage, liposome fusion, cell fusion, cell lysis, andendosomal release. Membrane active polymers that can cause lysis of cellmembranes are also termed membrane lytic polymers. Polymers thatpreferentially cause disruption of endosomes or lysosomes over plasmamembrane are considered endosomolytic. The effect of membrane activepolymers on a cell membrane may be transient. Membrane active polymerspossess affinity for the membrane and cause a denaturation ordeformation of bilayer structures. Membrane active polymers may besynthetic or non-natural amphipathic polymers.

Delivery of a polynucleotide to a cell is mediated by the membraneactive polymer disrupting or destabilizing the plasma membrane or aninternal vesicle membrane (such as an endosome or lysosome), includingforming a pore in the membrane, or disrupting endosomal or lysosomalvesicles thereby permitting release of the contents of the vesicle intothe cell cytoplasm.

Endosomolytic

Endosomolytic polymers are polymers that, in response to a change in pH,are able to cause disruption or lysis of an endosome or provide forrelease of a normally cell membrane impermeable compound, such as apolynucleotide or protein, from a cellular internal membrane-enclosedvesicle, such as an endosome or lysosome. Endosomolytic polymers undergoa shift in their physico-chemical properties over a physiologicallyrelevant pH range (usually pH 5.5-8). This shift can be a change in thepolymer's solubility or ability to interact with other compounds ormembranes as a result in a shift in charge, hydrophobicity, orhydrophilicity. Exemplary endosomolytic polymers have pH-labile groupsor bonds. A reversibly masked membrane active poly(vinyl ester), whereinthe masking agents are attached to the polymer via pH labile bonds, cantherefore be considered to be an endosomolytic polymer.

Hydrophilic Group

Hydrophilic group indicates in qualitative terms that the chemical groupis water-preferring. Typically, such chemical groups are water soluble,and are hydrogen bond donors or acceptors with water. A hydrophilicgroup can be charged or uncharged. Charged groups can be positivelycharged (anionic) or negatively charged (cationic) or both(zwitterionic). Examples of hydrophilic groups include the followingchemical moieties: carbohydrates, polyoxyethylene, certain peptides,oligonucleotides, amines, amides, alkoxy amides, carboxylic acids,sulfurs, and hydroxyls.

Hydrophobic Group

Hydrophobic group indicates in qualitative terms that the chemical groupis water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to form hydrogen bonds. Hydrophobic groupsdissolve in fats, oils, lipids, and non-polar solvents and have littleto no capacity to form hydrogen bonds. Hydrocarbons containing two (2)or more carbon atoms, certain substituted hydrocarbons, cholesterol, andcholesterol derivatives are examples of hydrophobic groups andcompounds.

Hydrophobic groups are preferably hydrocarbons, containing only carbonand hydrogen atoms. However, non-polar substitutions or non-polarheteroatoms which maintain hydrophobicity, and include, for examplefluorine, may be permitted. The term includes aliphatic groups, aromaticgroups, acyl groups, alkyl groups, alkenyl groups, alkynyl groups, arylgroups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each ofwhich may be linear, branched, or cyclic. The term hydrophobic groupalso includes: sterols, steroids, cholesterol, and steroid andcholesterol derivatives. As used herein, lower hydrophobic monomers orgroups comprise hydrophobic groups having two (2) to six (6) carbonatoms. As used herein, medium hydrophobic monomers or groups comprisehydrophobic groups having seven (7) to eleven (11) carbon atoms. As usedherein, higher hydrophobic monomers or groups comprise hydrophobicgroups having twelve (12) to thirty-six (36) or more carbon atoms.

Targeting Group

Targeting groups or moieties enhance the pharmacokinetic orbiodistribution properties of a conjugate to which they are attached toimprove cell-specific distribution and cell-specific uptake of theconjugate. Targeting groups enhance the association of molecules with atarget cell. Thus, targeting groups can enhance the pharmacokinetic orbiodistribution properties of a conjugate to which they are attached toimprove cellular distribution and cellular uptake of the conjugate.Binding of a targeting group, such as a ligand, to a cell or cellreceptor may initiate endocytosis. Targeting groups may be monovalent,divalent, trivalent, tetravalent, or have higher valency. Targetinggroups may be selected from the group comprising: compounds withaffinity to cell surface molecule, cell receptor ligands, andantibodies, antibody fragments, and antibody mimics with affinity tocell surface molecules. A preferred targeting group comprises a cellreceptor ligand. A variety of ligands have been used to target drugs andgenes to cells and to specific cellular receptors. Cell receptor ligandsmay be selected from the group comprising: carbohydrates, glycans,saccharides (including, but not limited to: galactose, galactosederivatives, mannose, and mannose derivatives), vitamins, folate,biotin, aptamers, and peptides (including, but not limited to:RGD-containing peptides, insulin, EGF, and transferrin).

ASGPr Targeting Group

Galactose and galactose derivates have been used to target molecules tohepatocytes in vivo through their binding to the asialoglycoproteinreceptor (ASGPr) expressed on the surface of hepatocytes. As usedherein, an ASGPr targeting group comprises a galactose and galactosederivative (structural analog) having affinity for the ASGPr equal to orgreater than that of galactose. Binding of galactose targeting moietiesto the ASGPr(s) facilitates cell-specific targeting of the deliverypolymer to hepatocytes and endocytosis of the delivery polymer intohepatocytes.

ASGPr targeting moieties may be selected from the group comprising:lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine,N-formylgalactosamine, N-acetyl-galactosamine, N-propionylgalactosamine,N-n-butanoylgalactosamine, N-iso-butanoyl-galactosamine,oligosaccharides, saccharide clusters (such as: Tyr-Glu-Glu-(aminohexylGalNAc)₃, lysine-based galactose clusters, and cholane-based galactoseclusters) (Iobst, S. T. and Drickamer, K. J.B.C. 1996, 271, 6686). ASGPrtargeting moieties can be monomeric (e.g., having a singlegalactosamine) or multimeric (e.g., having multiple galactosamines).Further suitable conjugates can include oligosaccharides that can bindto carbohydrate recognition domains (CRD) found on theasialoglycoprotein-receptor (ASGP-R). Example conjugate moietiescontaining oligosaccharides and/or carbohydrate complexes are providedin U.S. Pat. No. 6,525,031.

In some embodiments, an ASGPr targeting group is linked to anamine-reactive group, such as a maleic anhydride, through a PEG linkeras illustrated by the structure:

wherein n is an integer between 1 and 19 (inclusive).

In one embodiment, an ASGPr targeting group comprises a galactosecluster (galactose cluster targeting group). As used herein, a galactosecluster comprises a molecule having two to four terminal galactosederivatives. A terminal galactose derivative is attached to a moleculethrough its C-1 carbon. A preferred galactose cluster has three terminalgalactosamines or galactosamine derivatives each having affinity for theasialoglycoprotein receptor. A more preferred galactose cluster hasthree terminal N-acetyl-galactosamines. Other terms common in the artinclude tri-antennary galactose, tri-valent galactose and galactosetrimer. It is known that tri-antennary galactose derivative clusters arebound to the ASGPr with greater affinity than bi-antennary ormono-antennary galactose derivative structures (Baenziger and Fiete,1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257,939-945).

A galactose cluster contains three galactose derivatives each linked toa central branch point. The galactose derivatives are attached to thecentral branch point through the C-1 carbons of the saccharides. Thegalactose derivative is preferably linked to the branch point vialinkers or spacers. A preferred spacer is a flexible hydrophilic spacer(U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p.1538-1546). A preferred flexible hydrophilic spacer is a PEG spacer. Apreferred PEG spacer is a PEG_(S) spacer. The branch point can be anysmall molecule which permits attachment of the three galactosederivatives and further permits attachment of the branch point to theRNAi polynucleotide. An exemplary branch point group is a di-lysine. Adi-lysine molecule contains three amine groups through which threegalactose derivatives may be attached and a carboxyl reactive groupthrough which the di-lysine may be attached to the RNAi polynucleotide.

Galactose Cluster with PEG Spacer between Branch Point and Nucleic AcidSteric Stabilizer

As used herein, a steric stabilizer is a non-ionic hydrophilic polymer(either natural, synthetic, or non-natural) that prevents or inhibitsintramolecular or intermolecular interactions of a polymer to which itis attached relative to the polymer containing no steric stabilizer. Asteric stabilizer hinders a polymer to which it is attached fromengaging in electrostatic interactions. Electrostatic interaction is thenon-covalent association of two or more substances due to attractiveforces between positive and negative charges. Steric stabilizers caninhibit interaction with blood components and therefore opsonization,phagocytosis, and uptake by the reticuloendothelial system. Stericstabilizers can thus increase circulation time of molecules to whichthey are attached. Steric stabilizers can also inhibit aggregation of apolymer. A preferred steric stabilizer is a polyethylene glycol (PEG) orPEG derivative. As used herein, a preferred PEG can have about 1-500ethylene glycol monomers, 2-20 ethylene glycol monomers, 5-15 ethyleneglycol monomers, or about 10 ethylene glycol monomers. As used herein, apreferred PEG can also have a molecular weight average of about85-20,000 Daltons (Da), about 200-1000 Da, about 200-750 Da, or about550 Da. As used herein, steric stabilizers prevent or inhibitintramolecular or intermolecular interactions of a polymer to which itis attached relative to the polymer containing no steric stabilizer inaqueous solution.

A structural analog is a compound having a structure similar to that ofanother one, but differing from it in respect of a certain component. Itcan differ in one or more atoms, functional groups, or substructures,which are replaced with other atoms, groups, or substructures.Typically, a structural analog differs in the replacement of a singleelement, i.e. replacement of one atom or functional group by anotheratom of a different element or functional group. A structural analog canbe imagined to be formed, at least theoretically, from the othercompound. As such, a structural analog has a high chemical similarity tothe other compound. As typically used in the art, despite structuralsimilarity, structural analogs may have very different physical,chemical, or biochemical properties. However, as used herein withrespect to their stated properties, structural analogs have similarphysical, chemical, or biochemical properties.

Surface Charge

Zeta potential is a physical property which is exhibited by a particlein suspension and is closely related to surface charge. In aqueousmedia, the pH of the sample is one of the most important factors thataffects zeta potential. When charge is based uponprotonation/deprotonation of bases/acids, the charge is dependent on pH.Therefore, a zeta potential value must include the solution conditions,especially pH, to be meaningful. For typical particles, the magnitude ofthe zeta potential gives an indication of the potential stability of thecolloidal system. If all the particles in suspension have a largenegative or positive zeta potential, they will tend to repel each otherand there will be no tendency for the particles to come together.However, if the particles have low zeta potential values, there will beno force to prevent the particles coming together and flocculating. Thegeneral dividing line between stable and unstable suspensions fortypical particles is generally taken at either +30 or −30 mV. Particleswith zeta potentials more positive than +30 mV or more negative than −30mV are normally considered stable. Delivery polymers of the describedinvention exhibit a zeta potential of 20 mV to −20 mV at physiologicalsalt and pH 8, but are colloidally stable in aqueous solution and do notflocculate.

Positive charge, or zeta potential, of a membrane active polyamine isreduced by modification with the masking agents. Polymer charge,especially positive charge, can result in unwanted interactions withserum components or non-target cells. Positive surface charge also playsa role in membrane activity by enhancing interaction of the polymer withnegatively charged cell membranes. Therefore, in vivo siRNA deliveryvehicles with near neutral net charge or zeta potential are preferred.Delivery polymers of the invention, membrane active polyamines modifiedby reversible attachment of ASGPr targeting group masking agents andsteric stabilizer masking agents, have an apparent surface charge nearneutral and are serum stable. More specifically, the delivery polymersof the invention have a zeta potential, measured at pH 8, between +30and −30 mV, between +20 and −20 mV, between +10 and −10 mV, or between+5 and −5 mV. At pH 7, the net charge of the conjugate is expected to bemore positive than at pH 8. Net charge, or surface charge, is asignificant factor for in vivo applications.

Labile Linkage

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. For example, alinkage can connect a modifying or masking agent to a polymer. Formationof a linkage may connect two separate molecules into a single moleculeor it may connect two atoms in the same molecule. The linkage may becharge neutral or may bear a positive or negative charge. A reversibleor labile linkage contains a reversible or labile bond. A linkage mayoptionally include a spacer that increases the distance between the twojoined atoms. A spacer may further add flexibility and/or length to thelinkage. Spacers may include, but are not be limited to, alkyl groups,alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenylgroups, aralkynyl groups; each of which can contain one or moreheteroatoms, heterocycles, amino acids, nucleotides, and saccharides.Spacer groups are well known in the art and the preceding list is notmeant to limit the scope of the invention.

A reversible or labile bond is a covalent bond other than a covalentbond to a hydrogen atom that is capable of being selectively broken orcleaved under conditions that will not break or cleave other covalentbonds in the same molecule. More specifically, a reversible or labilebond is a covalent bond that is less stable (thermodynamically) or morerapidly broken (kinetically) under appropriate conditions than othernon-labile covalent bonds in the same molecule. Cleavage of a labilebond within a molecule may result in the formation of two molecules. Forthose skilled in the art, cleavage or lability of a bond is generallydiscussed in terms of half-life (t_(1/2)) of bond cleavage (the timerequired for half of the bonds to cleave). Thus, reversible or labilebonds encompass bonds that can be selectively cleaved more rapidly thanother bonds in a molecule.

Appropriate conditions are determined by the type of labile bond and arewell known in organic chemistry. A labile bond can be sensitive to pH,oxidative or reductive conditions or agents, temperature, saltconcentration, the presence of an enzyme (such as esterases, includingnucleases, and proteases), or the presence of an added agent. Forexample, increased or decreased pH is the appropriate conditions for apH-labile bond.

The rate at which a labile group will undergo transformation can becontrolled by altering the chemical constituents of the moleculecontaining the labile group. For example, addition of particularchemical moieties (e.g., electron acceptors or donors) near the labilegroup can affect the particular conditions (e.g., pH) under whichchemical transformation will occur.

As used herein, a physiologically labile bond is a labile bond that iscleavable under conditions normally encountered or analogous to thoseencountered within a mammalian body. Physiologically labile linkagegroups are selected such that they undergo a chemical transformation(e.g., cleavage) when present in certain physiological conditions.

As used herein, a cellular physiologically labile bond is a labile bondthat is cleavable under mammalian intracellular conditions. Mammalianintracellular conditions include chemical conditions such as pH,temperature, oxidative or reductive conditions or agents, and saltconcentration found in or analogous to those encountered in mammaliancells. Mammalian intracellular conditions also include the presence ofenzymatic activity normally present in a mammalian cell such as fromproteolytic or hydrolytic enzymes. Physiologically labile bonds that arecleaved under appropriate conditions with a half-life of less than 45min. are considered very labile. Physiologically labile bonds that arecleaved under appropriate conditions with a half-life of less than 15min are considered extremely labile.

Chemical transformation (cleavage of the labile bond) occurs when amolecule containing the labile bond reaches an appropriate intra-and/orextra-cellular environment. For example, a pH labile bond may be cleavedwhen the molecule enters an acidified endosome. Thus, a pH labile bondmay be considered to be an endosomal cleavable bond. Enzyme cleavablebonds may be cleaved when exposed to enzymes such as those present in anendosome or lysosome or in the cytoplasm. A disulfide bond may becleaved when the molecule enters the more reducing environment of thecell cytoplasm. Thus, a disulfide may be considered to be a cytoplasmiccleavable bond.

As used herein, a pH-labile bond is a labile bond that is selectivelybroken under acidic conditions (pH<7). Such bonds may also be termedendosomally labile bonds, since cell endosomes and lysosomes have a pHless than 7. The term pH-labile includes bonds that are pH-labile, verypH-labile, and extremely pH-labile.

Reaction of an Amine with a Cyclic Anhydride to Form an Amide Acid

Cleavage of the amide acid to form an amine and an anhydride ispH-dependent and is greatly accelerated at acidic pH. This pH-dependentreactivity can be exploited to form reversible pH-labile bonds andlinkers.

Very pH-labile bond: A very pH-labile bond has a half-life for cleavageat pH 5 of less than 45 min. The construction of very pH-labile bonds iswell-known in the chemical art.

Extremely pH-labile bonds: An extremely pH-labile bond has a half-lifefor cleavage at pH 5 of less than 15 min. The construction of extremelypH-labile bonds is well-known in the chemical art.

Disubstituted cyclic anhydrides are particularly useful for modificationor attachment of masking agents to membrane active poly(vinyl ester)polymers of the invention. They provide physiologically pH-labilelinkages, readily modify amines, and restore those amines upon cleavagein the reduced pH found in cellular endosomes and lysosome. Second, theα or β carboxylic acid group created upon reaction with an amine,appears to contribute only about 1/20^(th) of the expected negativecharge to the polymer (Rozema et al. Bioconjugate Chemistry 2003). Thus,modification of the polyamine with the disubstituted maleic anhydrideseffectively neutralizes the positive charge of the polyamine rather thancreates a polymer with high negative charge. Near neutral polymers arepreferred for in vivo delivery.

RNAi Polynucleotide-Polynucleotide Targeting Group Conjugate

The RNAi polynucleotide-polynucleotide targeting group conjugate isformed by covalently linking the RNAi polynucleotide to thepolynucleotide targeting group. The polynucleotide is synthesized ormodified such that it contains a reactive group A. The polynucleotidetargeting group is also synthesized or modified such that it contains areactive group B. Reactive groups A and B are chosen such that they canbe linked via a covalent linkage using methods known in the art.

The polynucleotide targeting group may be linked to the 3′ or the 5′ endof the RNAi polynucleotide. For siRNA polynucleotides, the targetinggroup may be linked to either the sense strand or the antisense strand,though the sense strand is preferred.

In one embodiment, the polynucleotide targeting group consists of ahydrophobic group. More specifically, the polynucleotide targeting groupconsists of a hydrophobic group having at least 20 carbon atoms.Hydrophobic groups used as polynucleotide targeting moieties are hereinreferred to as hydrophobic targeting moieties. Exemplary suitablehydrophobic groups may be selected from the group comprising:cholesterol, dicholesterol, tocopherol, ditocopherol, didecyl,didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoid, andcholeamide.

The hydrophobic targeting group may be attached to the 3′ or 5′ end ofthe RNAi polynucleotide using methods known in the art. For RNAipolynucleotides having two strands, such as siRNA, the hydrophobic groupmay be attached to either strand.

The galactose cluster may be attached to the 3′ or 5′ end of the RNAipolynucleotide using methods known in the art. For RNAi polynucleotideshaving two strands, such as siRNA, the galactose cluster may be attachedto either strand.

Polynucleotide

The term polynucleotide, or nucleic acid or polynucleic acid, is a termof the art that refers to a polymer containing at least two nucleotides.Nucleotides are the monomeric units of polynucleotide polymers.Polynucleotides with less than 120 monomeric units are often calledoligonucleotides. Natural nucleic acids have a deoxyribose- orribose-phosphate backbone. A non-natural or synthetic polynucleotide isa polynucleotide that is polymerized in vitro or in a cell free systemand contains the same or similar bases but may contain a backbone of atype other than the natural ribose or deoxyribose-phosphate backbone.Polynucleotides can be synthesized using any known technique in the art.Polynucleotide backbones known in the art include: PNAs (peptide nucleicacids), phosphorothioates, phosphorodiamidates, morpholinos, and othervariants of the phosphate backbone of native nucleic acids. Basesinclude purines and pyrimidines, which further include the naturalcompounds adenine, thymine, guanine, cytosine, uracil, inosine, andnatural analogs. Synthetic derivatives of purines and pyrimidinesinclude, but are not limited to, modifications which place new reactivegroups on the nucleotide such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides. The term base encompasses any ofthe known base analogs of DNA and RNA. A polynucleotide may containribonucleotides, deoxyribonucleotides, synthetic nucleotides, or anysuitable combination. Polynucleotides may be polymerized in vitro, theymay be recombinant, contain chimeric sequences, or derivatives of thesegroups. A polynucleotide may include a terminal cap group at the 5′-end, the 3′ -end, or both the 5′ and 3′ ends. The cap group can be, butis not limited to, an inverted deoxy abasic group, an inverted deoxythymidine group, a thymidine group, or 3′ glyceryl modification.

An RNA interference (RNAi) polynucleotide is a molecule capable ofinducing RNA interference through interaction with the RNA interferencepathway machinery of mammalian cells to degrade or inhibit translationof messenger RNA (mRNA) transcripts of a transgene in a sequencespecific manner. Two primary RNAi polynucleotides are small (or short)interfering RNAs (siRNAs) and micro RNAs (miRNAs). RNAi polynucleotidesmay be selected from the group comprising: siRNA, miRNA, double-strandRNA (d5RNA), short hairpin RNA (shRNA), and expression cassettesencoding RNA capable of inducing RNA interference. siRNA comprises adouble stranded structure typically containing 15-50 base pairs andpreferably 21-25 base pairs and having a nucleotide sequence identical(perfectly complementary) or nearly identical (partially complementary)to a coding sequence in an expressed target gene or RNA within the cell.An siRNA may have dinucleotide 3′ overhangs. An siRNA may be composed oftwo annealed polynucleotides or a single polynucleotide that forms ahairpin structure. An siRNA molecule of the invention comprises a senseregion and an antisense region. In one embodiment, the siRNA of theconjugate is assembled from two oligonucleotide fragments wherein onefragment comprises the nucleotide sequence of the antisense strand ofthe siRNA molecule and a second fragment comprises nucleotide sequenceof the sense region of the siRNA molecule. In another embodiment, thesense strand is connected to the antisense strand via a linker molecule,such as a polynucleotide linker or a non-nucleotide linker. MicroRNAs(miRNAs) are small noncoding RNA gene products about 22 nucleotides longthat direct destruction or translational repression of their mRNAtargets. If the complementarity between the miRNA and the target mRNA ispartial, translation of the target mRNA is repressed. If complementarityis extensive, the target mRNA is cleaved. For miRNAs, the complex bindsto target sites usually located in the 3′ UTR of mRNAs that typicallyshare only partial homology with the miRNA. A “seed region” —a stretchof about seven (7) consecutive nucleotides on the 5′ end of the miRNAthat forms perfect base pairing with its target—plays a key role inmiRNA specificity. Binding of the RISC/miRNA complex to the mRNA canlead to either the repression of protein translation or cleavage anddegradation of the mRNA. Recent data indicate that mRNA cleavage happenspreferentially if there is perfect homology along the whole length ofthe miRNA and its target instead of showing perfect base-pairing only inthe seed region (Pillai et al. 2007).

RNAi polynucleotide expression cassettes can be transcribed in the cellto produce small hairpin RNAs that can function as siRNA, separate senseand anti-sense strand linear siRNAs, or miRNA. RNA polymerase IIItranscribed DNAs contain promoters selected from the list comprising: U6promoters, H1 promoters, and tRNA promoters. RNA polymerase II promotersinclude U1, U2, U4, and U5 promoters, snRNA promoters, microRNApromoters, and mRNA promoters.

Lists of known miRNA sequences can be found in databases maintained byresearch organizations such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs (Pei et al. 2006, Reynolds et al. 2004,Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale etal. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

The polynucleotides of the invention can be chemically modified.Non-limiting examples of such chemical modifications include:phosphorothioate internucleotide linkages, 2′-O-methyl ribonucleotides,2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universalbase” nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasicresidue incorporation. These chemical modifications, when used invarious polynucleotide constructs, are shown to preserve polynucleotideactivity in cells while at the same time increasing the serum stabilityof these compounds. Chemically modified siRNA can also minimize thepossibility of activating interferon activity in humans.

In one embodiment, a chemically-modified RNAi polynucleotide of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is about 19 to about 29nucleotides. In one embodiment, an RNAi polynucleotide of the inventioncomprises one or more modified nucleotides while maintaining the abilityto mediate RNAi inside a cell or reconstituted in vitro system. An RNAipolynucleotide can be modified wherein the chemical modificationcomprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) of the nucleotides. An RNAi polynucleotide of the invention cancomprise modified nucleotides as a percentage of the total number ofnucleotides present in the RNAi polynucleotide. As such, an RNAipolynucleotide of the invention can generally comprise modifiednucleotides from about 5 to about 100% of the nucleotide positions(e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotide positions). Theactual percentage of modified nucleotides present in a given RNAipolynucleotide depends on the total number of nucleotides present in theRNAi polynucleotide. If the RNAi polynucleotide is single stranded, thepercent modification can be based upon the total number of nucleotidespresent in the single stranded RNAi polynucleotide. Likewise, if theRNAi polynucleotide is double stranded, the percent modification can bebased upon the total number of nucleotides present in the sense strand,antisense strand, or both the sense and antisense strands. In addition,the actual percentage of modified nucleotides present in a given RNAipolynucleotide can also depend on the total number of purine andpyrimidine nucleotides present in the RNAi polynucleotide. For example,wherein all pyrimidine nucleotides and/or all purine nucleotides presentin the RNAi polynucleotide are modified.

An RNAi polynucleotide modulates expression of RNA encoded by a gene.Because multiple genes can share some degree of sequence homology witheach other, an RNAi polynucleotide can be designed to target a class ofgenes with sufficient sequence homology. Thus, an RNAi polynucleotidecan contain a sequence that has complementarity to sequences that areshared amongst different gene targets or are unique for a specific genetarget. Therefore, the RNAi polynucleotide can be designed to targetconserved regions of an RNA sequence having homology between severalgenes thereby targeting several genes in a gene family (e.g., differentgene isoforms, splice variants, mutant genes, etc.). In anotherembodiment, the RNAi polynucleotide can be designed to target a sequencethat is unique to a specific RNA sequence of a single gene.

The term complementarity refers to the ability of a polynucleotide toform hydrogen bonds with another polynucleotide sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe polynucleotide molecules of the present invention, the binding freeenergy for a polynucleotide molecule with its target (effector bindingsite) or complementary sequence is sufficient to allow the relevantfunction of the polynucleotide to proceed, e.g., enzymatic mRNA cleavageor translation inhibition. Determination of binding free energies fornucleic acid molecules is well known in the art (Frier et al. 1986,Turner et al. 1987). A percent complementarity indicates the percentageof bases, in a contiguous strand, in a first polynucleotide moleculewhich can form hydrogen bonds (e.g., Watson-Crick base pairing) with asecond polynucleotide sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfectlycomplementary means that all the bases in a contiguous strand of apolynucleotide sequence will hydrogen bond with the same number ofcontiguous bases in a second polynucleotide sequence.

By inhibit, down-regulate, or knockdown gene expression, it is meantthat the expression of the gene, as measured by the level of RNAtranscribed from the gene or the level of polypeptide, protein, orprotein subunit translated from the RNA, is reduced below that observedin the absence of the blocking polynucleotide-conjugates of theinvention. Inhibition, down-regulation, or knockdown of gene expression,with a polynucleotide delivered by the compositions of the invention, ispreferably below that level observed in the presence of a controlinactive nucleic acid, a nucleic acid with scrambled sequence or withinactivating mismatches, or in absence of conjugation of thepolynucleotide to the masked polymer.

In Vivo Administration

In pharmacology and toxicology, a route of administration is the path bywhich a drug, fluid, poison, or other substance is brought into contactwith the body. In general, methods of administering drugs and nucleicacids for treatment of a mammal are well known in the art and can beapplied to administration of the compositions of the invention. Thecompounds of the present invention can be administered via any suitableroute, most preferably parenterally, in a preparation appropriatelytailored to that route. Thus, the compounds of the present invention canbe administered by injection, for example, intravenously,intramuscularly, intracutaneously, subcutaneously, or intraperitoneally.Accordingly, the present invention also provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient.

Parenteral routes of administration include intravascular (intravenous,intraarterial), intramuscular, intraparenchymal, intradermal, subdermal,subcutaneous, intratumor, intraperitoneal, intrathecal, subdural,epidural, and intralymphatic injections that use a syringe and a needleor catheter. Intravascular herein means within a tubular structurecalled a vessel that is connected to a tissue or organ within the body.Within the cavity of the tubular structure, a bodily fluid flows to orfrom the body part. Examples of bodily fluid include blood,cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of vesselsinclude arteries, arterioles, capillaries, venules, sinusoids, veins,lymphatics, bile ducts, and ducts of the salivary or other exocrineglands. The intravascular route includes delivery through the bloodvessels such as an artery or a vein. The blood circulatory systemprovides systemic spread of the pharmaceutical.

The described compositions are injected in pharmaceutically acceptablecarrier solutions. Pharmaceutically acceptable refers to thoseproperties and/or substances which are acceptable to the mammal from apharmacological/toxicological point of view. The phrase pharmaceuticallyacceptable refers to molecular entities, compositions, and propertiesthat are physiologically tolerable and do not typically produce anallergic or other untoward or toxic reaction when administered to amammal. Preferably, as used herein, the term pharmaceutically acceptablemeans approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals and more particularly inhumans.

Therapeutic Effect

RNAi polynucleotides may be delivered for research purposes or toproduce a change in a cell that is therapeutic. In vivo delivery of RNAipolynucleotides is useful for research reagents and for a variety oftherapeutic, diagnostic, target validation, genomic discovery, geneticengineering, and pharmacogenomic applications. We have disclosed RNAipolynucleotide delivery resulting in inhibition of endogenous geneexpression in hepatocytes. Levels of a reporter (marker) gene expressionmeasured following delivery of a polynucleotide indicate a reasonableexpectation of similar levels of gene expression following delivery ofother polynucleotides. Levels of treatment considered beneficial by aperson having ordinary skill in the art differ from disease to disease.For example, Hemophilia A and B are caused by deficiencies of theX-linked clotting factors VIII and IX, respectively. Their clinicalcourse is greatly influenced by the percentage of normal serum levels offactor VIII or IX: <2%, severe; 2-5%, moderate; and 5-30% mild. Thus, anincrease from 1% to 2% of the normal level of circulating factor insevere patients can be considered beneficial. Levels greater than 6%prevent spontaneous bleeds but not those secondary to surgery or injury.Similarly, inhibition of a gene need not be 100% to provide atherapeutic benefit. A person having ordinary skill in the art of genetherapy would reasonably anticipate beneficial levels of expression of agene specific for a disease based upon sufficient levels of marker generesults. In the hemophilia example, if marker genes were expressed toyield a protein at a level comparable in volume to 2% of the normallevel of factor VIII, it can be reasonably expected that the gene codingfor factor VIII would also be expressed at similar levels. Thus,reporter or marker genes serve as useful paradigms for expression ofintracellular proteins in general.

The liver is an important target tissue for RNAi therapy given itscentral role in metabolism (e.g., lipoprotein metabolism in varioushypercholesterolemias) and the secretion of circulating proteins (e.g.,clotting factors in hemophilia). In addition, acquired disorders such aschronic hepatitis and cirrhosis are common and are also potentiallytreated by RNAi therapies. A number of diseases or conditions whichaffect or are affected by the liver are potentially treated throughknockdown (inhibition) of gene expression in the liver. Such liverdiseases and conditions may be selected from the list comprising: livercancers (including hepatocellular carcinoma, HCC), viral infections(including hepatitis), metabolic disorders, (including hyperlipidemiaand diabetes), fibrosis, and acute liver injury.

The amount (dose) of delivery polymer and RNAi-polynucleotide-conjugatethat is to be administered can be determined through routineexperimentation. We have shown effective knockdown of gene expressionusing 0.05-20 mg/kg animal weight of siRNA-conjugate and 1.5-60 mg/kganimal weight delivery polymer. A preferred amount in mice is 0.25-2.5mg/kg siRNA-conjugate and 1-40 mg/kg delivery polymer. More preferably,about 2-20 mg/kg delivery polymer is administered.

As used herein, in vivo means that which takes place inside an organismand more specifically to a process performed in or on the living tissueof a whole, living multicellular organism (animal), such as a mammal, asopposed to a partial or dead one.

Transfection Reagent

The poly(vinyl ester)s described herein may be used as in vitrotransfection reagents. The process of delivering a polynucleotide to acell in vitro has been commonly termed transfection or the process oftransfecting. The term transfecting as used herein refers to theintroduction of a polynucleotide from outside a cell to inside the cellsuch the polynucleotide has biological activity. The polynucleotide maybe used for research purposes or to produce a change in a cell that canbe therapeutic. The delivery of a polynucleotide can lead tomodification of the genetic material present in the target cell.

An in vitro transfection reagent is a compound or composition ofcompounds that binds to or complexes with oligonucleotides orpolynucleotides and mediates their entry into a cell, typically amammalian cell in vitro. Examples of in vitro transfection reagentsinclude, but are not limited to, protein and polymer complexes(polyplexes), lipids and liposomes (lipoplexes), combinations ofpolymers and lipids (lipopolyplexes), calcium phosphate precipitates,and dendrimers. Typically, the in vitro transfection reagent has acomponent with a net positive charge which associates or complexes with,via electrostatic interaction, the negative charge of theoligonucleotide or polynucleotide. Cationic in vitro transfection agentsmay also condense large nucleic acids. In addition to their utility forin vivo delivery, the poly(acrylate)s described herein can also be usedas in vitro transfection reagents. For use as in vitro transfectionreagents, the poly(acrylate)s may be masked or unmasked.

EXAMPLES Example 1 Synthesis of polymer monomers

A. Materials. Vinyl acetate (VAc), vinyl butyrate (VBu), Pd (II)acetate, γ-(Boc-amino)butyric acid (Boc-GABA), 5-(Boc-amino)valeric acid(Boc-5-Ava-OH), valeric acid, carbon disulfide, sodium hydride,tetrahydrofuran (THF), dimethylsulfoxide (DMSO), diphenylamine, diethylchloromalonate, and magnesium sulfate (MgSO₄) purchased fromSigma-Aldrich and used without further purification. Monomer3-tert-butoxycarbonylamino-propionic vinyl ester (BAPVE) was purchasedfrom Sigma-Aldrich prior to dissolving in ethyl acetate and passingthrough an alumina plug to remove inhibitor.

B. 4-tert-Butoxycarbonylamino-Butyric Acid Vinyl Ester (BABVE) 3protected amine vinyl ester monomer. γ-(Boc-amino)butyric acid 2(Boc-GABA, 10 g, 49.20 mmol, CAS 57294-38-9) was dissolved in vinylacetate 1 (450 mL, 4920 mmol, CAS 108-05-4) at room temperature (RT).Once dissolved, Pd (II) acetate (2.21 g, 9.84 mmol, CAS 3375-31-3) andKOH (276 mg, 4.92 mmol, CAS 1310-58-3) were added, and the reactionmixture was stirred overnight at RT. The reaction mixture was thentransferred into a large excess of diethyl ether to precipitate black Pdbyproduct. The solution plus precipitate was then filtered throughcelite to remove the black precipitate. The resulting solution was thenconcentrated to dryness and the product 3 was purified on a silicacolumn using 15% ethyl acetate in hexane eluent. Typical yield was70-90%. Molecular weight: 229.28.

C. 5-tent-Butoxycarbonylamino-Valeric Acid Vinyl Ester (BAVVE) 6protected amine monomer. 5-(Boc-amino)valeric acid 5 (Boc-5-Ava-OH, 10g, 46.03 mmol, CAS 27219-07-4) was dissolved in vinyl acetate 1 (424 mL,4603 mmol) at RT. Once dissolved, Pd (II) acetate (2.07 g, 9.21 mmol)and KOH (258 mg, 4.60 mmol) were added and the reaction mixture wasstirred overnight at RT. The reaction mixture was then transferred intoa large excess of diethyl ether to fully precipitate the black Pdbyproduct. The solution plus precipitate was then filtered throughcelite to remove the black precipitate. The resulting solution was thenconcentrated to dryness and the product 6 was purified on a silicacolumn using ethyl acetate/hexane eluent. Typical yield was 70-90%.Molecular weight 243.31.

D. Vinyl Valerate (VV) 8 hydrophobic monomer (CAS 5873-43-8). Valericacid 7 (5 g, 48.96 mmol, CAS 109-52-4) was dissolved in vinyl acetate 1(450 mL, 4896 mmol) at RT.

Once dissolved, Pd(II) acetate (2.20 g, 9.79 mmol) and KOH (275 mg, 4.89mmol) were added, and the reaction mixture was stirred overnight at RT.The reaction mixture was then transferred into a large excess of diethylether to fully precipitate a black Pd compound. The solution plusprecipitate was then filtered through celite to remove the blackprecipitate. The resulting solution was then concentrated to dryness andthe product was purified on a silica column using ethyl acetate/hexaneeluent. Typical yield was about 30%. Molecular weight 128.17.

E. Synthesis of 3-(2-tert-butoxycarbonylamino-ethoxy)-propionic vinylester (BEPVE).

1) Synthesis of 3-(2-tent-Butoxycarbonylamino-ethoxy)-propionic acidtent-butyl ester. In a flame-dried round bottom flask purged with argon,Boc-ethanolamine (10 mL, 64.64 mmol) and tert-butyl acrylate (18.82 mL,129.28 mmol) were dissolved in dioxane (25 mL) and heated to 25° C. A60% KOH solution (1.5 mL) was added and the reaction mixture stirredovernight at 25° C. The reaction was monitored by TLC, and more KOHsolution was added until most of the starting Boc-ethanolamine wasconsumed. The reaction mixture was then mixed with DCM, and washed 3times with deionized water and once with brine. The organic layer wasrecovered, dried over Na₂SO₄, and the solvent removed via rotaryevaporation. The resulting oil was purified on a silica column using anethyl acetate/hexane eluent. Yield=70%.

2) 3-(2-Amino-ethoxy)-propionic acid.3-(2-tert-Butoxycarbonylamino-ethoxy)-propionic acid tert-butyl ester(10 g) was dissolved in a 1:1 trifluoroacetic acid (TFA)/DCM mixture(100 mL) and stirred at room temperature for 1 h. The solvent was thenremoved on a rotary evaporator and the resulting oil was re-dissolved inDCM and concentrated to dryness. This process was repeated until minimalTFA scent remained before the oil was dried on high vacuum for severalhours.

3) 3-(2-tert-Butoxycarbonylamino-ethoxy)-propionic acid.3-(2-Amino-ethoxy)-propionic acid was dissolved in a minimal amount ofdeionized water before the pH was raised to 8.5 by careful addition of 1M NaOH. After the pH adjustment, Boc₂O (1 M in THF, 2 eq.) was addeddropwise to the solution which was stirred overnight at roomtemperature. THF was removed by rotary evaporation and reaction mixturedissolved in 1:1 ethyl acetate/methanol and washed with 10% citric acidsolution. The aqueous layer was extracted three times with ethylacetate, and two times with DCM. The organic layers were combined, driedover Na₂SO₄, and concentrated to dryness under vacuum. The resultingcrude oil was purified by silica gel chromatography with an ethylacetate/hexane eluent. Yield=30%.

4) 3-(2-tert-Butoxycarbonylamino-ethoxy)-propionic vinyl ester.3-(2-tert-Butoxycarbonylamino-ethoxy)-propionic acid (3.72 g, 15.95mmol) was dissolved in vinyl acetate (147 mL, 1595 mmol). Pd (II)acetate (716 mg, 3.19 mmol) and KOH (89 mg, 1.59 mmol) were added to thereaction mixture and stirred overnight. The reaction mixture wastransferred into a large excess of diethyl ether to fully precipitatethe black Pd byproduct. The solution plus precipitate was then filteredthrough celite to remove the black precipitate. The resulting solutionwas then concentrated to dryness and the product purified on a silicacolumn using ethyl acetate/hexane eluent. Yield=60%.

F. Synthesis of malonate N,N-diphenyl dithiocarbamate (MDPD). MDPD wassynthesized according to a procedure described by Shipp et al. Briefly,NaH (1.24 g, 0.0520 mol) was suspended in THF (10 mL) and cooled to 0°C. using an ice bath. A solution of diphenylamine (3.38 g, 0.0200 mol)in DMSO (18 mL) and THF (9 mL) was added and stirred for 1 h at 0° C.Carbon disulfide (2.84 mL, 0.0472 mol) was added and the solutionstirred for a further 30 min at 0° C. The solution was then cooled usingan ethylene glycol/CO2 bath prior to the addition of diethylchloromalonate (3.23 mL, 0.0200 mol) and further stirring for 2 h atroom temperature. Any remaining NaH was hydrolyzed with methanol and theproduct was extracted with diethyl ether. Volatiles were then removedand the product was purified using a silica column (ethyl acetate:hexanemix 10:90 to remove diphenylamine impurity, followed by 30:70 to eluteproduct). The product was dried under vacuum to yield a yellow solid(yield 72%).

Example 2 RAFT Copolymerization of Vinyl Ester Monomers to FormAmphiphilic Cationic Poly(vinyl ester) Random Copolymers

A. Reversible Addition-Fragmentation chain Transfer (RAFT)polymerizations were carried out according to Shipp et al. 2009 usingMalonate N,N-diphenyl dithiocarbamate 9 (MDP-DTC).

Vidyasagar Malepu et al. “RAFT Polymerization of Vinyl Acetate, Styreneand Acrylates Using N,N-Dithiocarbamates” in Controlled/Living RadicalPolymerization: Progress in RAFT, DT, NMP & OMRP, Matyjaszewski K,editor; ACS Symposium Series, Vol. 1024, chapter 3, pp 37-47; AmericanChemical Society, Washington D.C., 2009.

B. Polymer Calculations: polymer theoretical molecular weight(M_(n, th)), moles monomers, moles Chain Transfer Agent, molesInitiator.

General Reaction for Synthesis of Polymer P from Monomers A and B

-   -   A=Hydrophilic Monomer    -   B=Hydrophobic Monomer    -   P=Polymer    -   C=Chain Transfer Agent (CTA)    -   I=Initiator

Calculation of Monomer Average Molecular Weight for Polymer P[%A×MW _(A)]+[%B×MW _(B) ]= MW _(AB)

-   -   %A=percent hydrophilic monomers A in polymer P    -   %B=percent hydrophobic monomers B in polymer P    -   MW_(A)=Molecular weight of hydrophilic monomer A    -   MW_(B)=Molecular weight of hydrophobic monomer B    -   MW _(AB)=Average molecular weight of polymer monomers

Calculation of number of monomers in polymer P having a desired(theoretical) molecular weight M_(n, th) (M_(n) in the equation below):M _(n) / MW _(AB) =n _(AB)

-   -   n_(AB)=number of monomers in polymer P having theoretical        molecular weight M_(n, th)

Calculation of moles of monomers A and B in x grams polymer P havingtheoretical molecular weight M_(n, th):

$\left\lbrack {\%\mspace{14mu} A \times n_{AB} \times \left( \frac{x}{M_{n}} \right)} \right\rbrack = {{{moles}_{A}\left\lbrack {\%\mspace{14mu} B \times n_{AB} \times \left( \frac{x}{M_{n}} \right)} \right\rbrack} = {moles}_{B}}$

-   -   moles_(A)=moles hydrophilic monomer A    -   moles_(B)=moles hydrophobic monomer B

Calculation of moles Chain Transfer Agent for synthesis of x gramspolymer P having theoretical molecular weight M_(n, th):moles_(A) /[n _(AB)×%A]=moles_(B) /[n _(AB)×%B]=moles_(C)

-   -   moles_(C)=moles Chain Transfer Agent

Calculation of moles Initiator for synthesis of x grams polymer P havingtheoretical molecular weight M_(n, th):%I×moles_(C)=moles_(I)

-   -   moles_(I)=moles Initiator

C. General procedure for RAFT polymerization of protected amine vinylester random copolymers. CTA and Initiator are combined in a reactionvessel dried under high vacuum. Hydrophilic monomer is added and themixture is degassed for 1 hour by N₂ bubbling. A separate vial of excesshydrophobic monomer is similarly degassed. A measured amount of degassedhydrophobic monomer is added to reaction vessel and the mixture isstirred at 95° C. overnight. After ˜16 hours, the reaction vessel isremoved from heat and the solution is allowed to cool to RT. Theresulting gel is dissolved in dichloromethane (DCM) and the polymer isprecipitated by addition of hexane (˜8× vol.). After centrifugation, thesolution is decanted and the polymer rinsed with hexane. The rinsedpolymer is redissolved in DCM, and precipitated again with hexane (˜8×vol.). After centrifugation, the solution is decanted and the polymerdried under high vacuum.

D. NMR Analysis. A sample of the polymer is prepared at 7.5 mg/ml inCDCl₃. A ¹H-NMR spectrum is taken on the Varian 400 Hz MR instrumentwith a 2 sec relaxation delay and 32-64 scans. The spectrum is analyzedwith manual phasing followed by integral and baseline corrections.

E. MALS (Multi-Angle Light Scattering) Molecular weight analysis. Asample of the polymer is brought up at 10 mg/ml in a 0.02 μm Whatmananodisc filtered buffer of DCN, 20% THF, 5% ACN. The solution is thenfiltered through a 0.1 μm Whatman anotop filter. The samples are run at0.75 ml/min in the above buffer through a Jordi Gel DVB mixed bedanalytical column. The sample is then passed through the HELEOS lightscattering detector and the optilab REX RI detector. The data iscollected and analyzed using ASTRA V software using a previouslydetermined dn/dc of 0.63 ml/gm. The ASTRA V analysis provides Mw andM_(n).

Example 3 Synthesis of Poly (5-tert-butoxycarbonylaminovaleric vinylester-co-vinyl butyrate), P(BAVVE-co-VBu)

A. Amine-protected DAN-41947-106 poly(vinyl ester) random copolymersynthesis. Malonate N,N-diphenyl dithiocarbamate (MDPC, 2.56 mg, 0.0066mmol) and benzoyl peroxide (BPO, 0.795 mg, 0.00328 mmol) were dried in areaction vessel and 5-tert-Butoxycarbonylamino-valeric vinyl ester 3(1.00 g, 4.11 mmol) was added. The mixture was degassed by N₂ bubblingfor 1 h. A separate vial of vinyl butyrate (VBu) was similarly degassed.VBu (345 μL, 2.74 mmol, CAS 123-20-6) was added to the reaction vesseland the mixture was stirred overnight at 95° C. After ˜16 h, thereaction vessel was removed from heat and the solution was allowed tocool to room temperature (RT). The resulting gel was dissolved in 5 mLDCM and the polymer was precipitated by addition of 40 mL hexane. Aftercentrifugation, the solution was decanted and the polymer was rinsedwith 5 mL hexane. The rinsed polymer was redissolved in 5 mL DCM, andprecipitated again with 40 mL hexane. After centrifugation, the solutionwas decanted and the polymer was dried under high vacuum. ¹H NMR(CDCl₃): δ 7.4, 4.7-5.25, 3.1, 2.15-2.4, 1.45-1.95, 0.95.

B. Precipitation of polymer. After drying, the polymer was dissolved inDCM to a concentration of 100 mg/mL and fully or fractionallyprecipitated by addition of hexane.

Full precipitation and fractional precipitation of (co)polymers. Afterpolymerization, the reaction solution was allowed to cool to roomtemperature and transferred to a 50 mL centrifuge tube. DCM (2 mL) wasused to wash out the reaction vessel and help transfer the reactionsolution before hexane (35 mL) was added to the solution. The solutionwas centrifuged for 2 min at 4,400 rpm. The supernatant layer wascarefully decanted and the bottom (viscous liquid or solid) layer wasrinsed with hexane. The bottom layer was re-dissolved in DCM (7 mL),precipitated in hexane (35 mL), and centrifuged once more. Thesupernatant was decanted and the bottom layer rinsed with hexane beforethe polymer was dried under reduced pressure for several hours.

Fractional precipitation of (co)polymers. After polymerization, thereaction solution was allowed to cool to room temperature andtransferred to a 50 mL centrifuge tube. DCM (2 mL) was used to wash outthe reaction vessel and help transfer the reaction solution beforehexane (35 mL) was added to the solution. The solution was centrifugedfor 2 min at 4,400 rpm. The supernatant layer was carefully decanted andthe bottom (viscous liquid or solid) layer was rinsed with hexane. Thebottom layer was re-dissolved in DCM (100 mg/mL polymer) before hexanewas added. In this case, enough hexane to precipitate half of the totalpolymer was added—typically, the amount of hexane required to take thesolution just past the cloud point. The amount of hexane added to reachthis point varied depending on the type and molecular weight of thecopolymer solution. The cloudy mixture was centrifuged (3 min at 4,400rpm), forming two liquid layers. The thicker bottom layer was removedusing a glass pipette, diluted with DCM (5 mL), and fully precipitatedby adding hexane (30 mL) to yield fraction 1. Hexane was added to thetop layer to make a total volume of 50 mL and fully precipitate fraction2. Both precipitates were centrifuged (2 min at 4,400 rpm), and thefractions recovered by decanting the supernatant, rinsing theprecipitated polymer with hexane, and finally dried under reducedpressure for several hours.

C. Polymer Deprotection. Dried polymer was dissolved in 5-10 mL of 2 MHCl in acetic acid solution and stirred at RT for 1 hour. The reactionmixture was diluted with 40 mL deionized H₂O (dH₂O), placed in adialysis bag with nominal MWCO of ˜3500 and dialyzed twice for 8-16hours in high salt (NaCl) and twice for 8-16 hours in dH₂O. The polymerwas then lyophilized.

Similar procedures were followed for the copolymerization of BAVVE withVAc, VPr, VBu, or VV. In some cases, 0-3 mL butyl acetate was added tothe reaction mixture prior to degassing. The relative concentration ofMDPD, initiator, and monomers were altered.

TABLE 1 Exemplary BAVVE-based poly(vinyl ester) copolymers. hydrophobicmonomer measured polymer monomer feed ratio M_(n, th) PDI DAN-41947-109propionyl 60/40 100 K DAN-41947-110 200 K DAN-41947-107 70/30 100 KDAN-41947-108 200 K DAN-42435-15-A-1 butyryl 56:44 200 KDAN-41947-47-A-1 60:40  30 K 1.23 DAN-41947-47-B-1  50 K 1.27DAN-41947-47-C-1  75 K 1.44 DAN-41947-47-D-1 100 K 1.51 DAN-41947-47-E-1150 K 1.63 DAN-41947-105 100 K DAN-41947-106 or 200 K DAN-41947-129 ADAN-41947-103 70/30 100 K DAN-41947-104 200 K DAN-42435-14-B-1 75:25 200K DAN-42435-14-A-1 80:20 200 K DAN-41947-123 A valeryl 60/40 100 KDAN-41947-123 B 200 K DAN-41947-122 A 70/30 100 K DAN-41947-122 B 200 KDAN-42435-78-A-1 hexanyl 60:40 200 K DAN-42435-80-A-1 octanyl 60:40 200K

Example 4 Synthesis of Poly (5-tert-butoxycarbonylaminoproprionic vinylester-co-vinylbutyrate), P(BAPVE-co-VBu)

A solution of malonate N,N-diphenyl dithiocarbamate (MDPD, 0.00876 g,0.0217 mmol), AIBN (0.89 mg, 0.00542 mmol), BAPVE (0.800 g, 0.00374mol), and butyl acetate (BuAc, 1 mL) were added to a 20 ml vial anddegassed by N₂ bubbling for 1 h. A separate vial of vinyl butyrate (VBu)was similarly degassed prior to addition via syringe (0.316 mL, 0.00249mmol). The mixture was stirred for 4 h at 80° C. and then allowed tocool to RT. The resulting viscous solution was dissolved in 5 mL DCM andthe polymer was precipitated by addition of 40 mL hexane. Aftercentrifugation, the upper solvent was decanted and the polymer wasrinsed with 5 mL hexane. The rinsed polymer was re-dissolved in 5 mLDCM, and precipitated once more with 40 mL hexane. After centrifugation,the upper solvent layer was decanted and the polymer was dried underhigh vacuum. ¹H NMR (CDCl₃): δ 7.4, 5.3-5.6, 4.7-5.1, 3.35, 2.5, 2.25,1.55-1.9, 1.45, 0.95. ¹³C NMR (CDCl₃): δ 171.5, 170.5, 155, 79, 66-68.5,39-41.5, 37, 35.5, 29.5, 19.5, 15. M_(n) 27,400 (M_(w)/M_(n) 1.34).Yield 74%.

TABLE 2 Exemplary BAPVE-based poly(vinyl ester) copolymers incor-hydrophobic monomer poration polymer monomer feed ratio ratio PDI Mn 32Aacetyl 60:40 60:40 1.52 29.8 K 32C 60:40 65:35 1.51 24.6 K 32D 70:3039:31 1.56 28.8 K 38A propionyl 60:40 66:34 1.47 21.6 K 38B 40:30 73:271.49 23.8 K DAN-42435-16-B-1 butyryl 60:40  200 K^(a) DAN-42435-16-A-170:30  200 K^(a)  3C 60:40 63:37 1.44 29.0 K 33A valeryl 60:40 68:321.57 24.5 K 33B 60:40 62:38 1.45 23.8 K ^(a)M_(n, th)

Similar procedures were followed for all copolymerizations of BAPVE withVAc, VPr, VBu, or VV. In some cases, 0-3 mL BuAc was added to thereaction mixture prior to degassing, while alternative initiators suchas BPO and ADMV were also used. The relative concentration of MDPD,initiator, and monomers were altered.

Example 5 Synthesis of Poly (4-tert-butoxycarbonylaminobutyric vinylester-co-vinyl butyrate), P(BABVE-co-VBu)

THF solutions of malonate N,N-diphenyl dithiocarbamate (MDPD, 2.56 mg,0.00634 mmol) and benzoyl peroxide (0.767 mg, 0.00317 mmol) were addedto a 20 mL vial and dried under vacuum for 30 min before BABVE (0.800mg, 0.00351 mol) was added. The mixture was degassed by N₂ bubbling for1 h. A separate vial of vinyl butyrate was similarly degassed. The vinylbutyrate (296 μL, 0.00234 mol) was added to the reaction vessel and themixture was stirred overnight at 95° C. After 16 h, the reaction vesselwas removed from heat and the solution was allowed to cool to RT. Theresulting gel was dissolved in 5 mL DCM and the polymer was precipitatedby addition of 40 mL hexane. After centrifugation, the solution wasdecanted and the polymer was rinsed with 5 mL hexane. The rinsed polymerwas redissolved in 5 mL DCM and precipitated once more with 40 mLhexane. After centrifugation, the solution was decanted and the polymerwas dried under high vacuum. ¹H NMR (CDCl₃): δ 7.4, 5.05-5.45, 4.7-5.05,3.15, 2.15-2.45, 1.55-1.9, 1.45, 0.95.

Similar procedures were followed for the copolymerization of BAPVE withVAc, VPr, VBu, or VV. In some cases, 0-3 mL butyl acetate was added tothe reaction mixture prior to degassing. The relative concentration ofMDPD, initiator, and monomers were altered.

TABLE 3 Exemplary BABVE-based poly(vinylester) copolymers. aminehydrophobic monomer polymer monomer monomer feed ratio M_(n, th)DAN-41947-90 butyric butyryl 50/50 100 K DAN-41947-93 60/40 100 KDAN-41947-96 200 K DAN-41947-89 70/30 100 K DAN-41947-95 200 KDAN-41947-115 A propionyl 60/40 100 K DAN-41947-115 B 200 KDAN-41947-114 A 70/30 100 K DAN-41947-114 B 200 K DAN-41947-119 Avaleryl 60/40 100 K DAN-41947-119 B 200 K DAN-41947-118 A 70/30 100 KDAN-41947-118 B 200 K

Example 6 Synthesis of poly3-(2-tent-butoxycarbonylaminoethoxy)propionic vinyl ester-co-vinylbutyrate), P(BEPVE-co-VBu)

A solution of malonate N,N-diphenyl dithiocarbamate (MDPD, 2.14 mg,0.0134 mmol), azobisisobutyronitrile (AIBN, 1.09 mg, 0.00665 mmol), andBEPVE (505 mg, 0.00214 mol) were added to a 20 mL vial and degassed byN₂ bubbling for 1 h. A separate vial of vinyl butyrate was similarlydegassed. The vinyl butyrate (162 mg, 0.00143 mol) was added to thereaction vessel and the mixture was stirred overnight at 95° C. After 16h, the reaction vessel was removed from heat and the solution wasallowed to cool to RT. The resulting viscous solution was dissolved in 5mL DCM and the polymer was precipitated by addition of 40 mL hexane.After centrifugation, the solution was decanted and the polymer wasrinsed with 5 mL hexane. The rinsed polymer was re-dissolved in 5 mL DCMand precipitated once more with 40 mL hexane. After centrifugation, thesolution was decanted and the polymer was dried under high vacuum. ¹HNMR (CDCl₃): δ 7.4, 5.1-5.5, 4.75-5.1, 3.65, 3.5, 3.28, 2.55, 2.25,1.55-1.9, 1.45, 0.95. ¹³C NMR (CDCl₃): δ 171.5, 169.5, 155, 79, 70.5,66.5, 41, 40, 37, 35.5, 29.5, 19.5, 15. Mn 23,100 (M_(w)/M_(n) 1.33).Yield 64%.

Similar procedures were followed for the copolymerization of BEPVE withVAc, VPr, VBu, or VV. In some cases, 0-3 mL butyl acetate was added tothe reaction mixture prior to degassing. The relative concentration ofMDPD, initiator, and monomers were altered.

Example 7 Masking Agents

A. Galactose Disubstituted Maleic Anhydride Masking Agents.

-   -   1) Compound 10

-   -    wherein        -   Y is neutral linker such as, but not limited to:            -   —(CH₂)_(a)—(O—CH₂—CH₂)_(b)—NH—CO—(CH₂)_(c)—, wherein a,                b and c are independently integers from 1-6, and        -   R is a galactose derivative having affinity for the            asialoglycoprotein receptor selected from the list            comprising:            -   OH (Galactose),            -   NH₂ (D-Galactosamine),            -   NH—CO—H (N-formyl-D-galactosamine),            -   NH—CO—CH₃ (N-acetyl-D-galactosamine (GalNAc)),            -   NH—CO—CH₂CH₃ (N-propionyl-D-galactosamine),            -   NH—CO—CH₂CH₂CH₃ (N-n-butanoyl-D-galactosamine), and            -   NH—CO—CH(CH₃)₂ (N-iso-butanoyl-D-galactosamine).

Reaction of the maleic anhydride with an anime group on the polymerresults in formation of a pH labile linkage between the galactose and apolymer amine.

-   -   2) Compound 11

-   -    wherein        -   Y is neutral linker such as, but not limited to:            —NH—(CH₂—CH₂—O)_(b)—(CH₂)_(a)—, wherein b and c are            independently integers from 1-6, and        -   R is as defined above for compound 10.    -   3) Compound 12

-   -    wherein n is an integer from 1 to 6 and R is as defined above        for compound 10.    -   4) Compound 13: N-Acetyl-galactosamine-PEG-methyl maleic        anhydride

-   -    wherein n is an integer from 1 to 6.    -   5) Alkyl spacer groups may also be used as illustrated in        compound 14.

-   -    wherein n is an integer from 0 to 10 and R is a defined above        for compound 10.

B. Polyethylene Glycol Disubstituted Maleic Anhydride masking agents.

-   -   1) Compound 15

-   -    wherein R is neutral and comprises a polyethylene glycol.

Reaction of the maleic anhydride with an anime group on the polymersresults in formation of a pH labile linkage between the PEG and apolymer amine.

-   -   2) Compound 16

-   -    wherein        -   n is an integer from 1 to 500, and        -   R is selected from the group consisting of —H, —CH₃, and            —CH₂—CH₃.

Preferably, n is an integer from 2 to 100. More preferably, the PEGcontains from 5 to 20 ethylene units (n is an integer from 5 to 20).More preferably, PEG contains 10-14 ethylene units (n is an integer from10 to 14). The PEG may be of variable length and have a mean length of5-20 or 10-14 ethylene units. Alternatively, the PEG may bemonodisperse, uniform or discrete; having, for example, exactly 11 or 13ethylene units.

C. Dipeptide masking agent, Compound 16

-   -   R1 and R2 are the R groups of amino acids,    -   R4 is a targeting ligand of a steric stabilizer,    -   X is —NH—, —O—, or —CH₂—,    -   Y is —NH— or —O—    -   R5 is at position 2, 4, or 6 and is —CH2—O—C(O)—O—Z wherein Z        carbonate, and    -   R6 is independently hydrogen, alkyl, or halide at each of        positions 2, 3, 4, 5, or 6 except for the position occupied by        R5.

Example 8 Conjugation of siRNA to Poly(vinyl ester) Random Copolymersvia Disulfide Bonds

Disulfide bonds can be made with varying kinetics of cleavage in thereducing environment in a typical mammalian cell.

A. SATA/SMPT linkage. N-succinimidyl-S-acetylthioacetate (SATA)-modifiedpolynucleotides were synthesized by reaction of 5′ amine-modified siRNAwith 1 weight equivalents (wt. eq.) of SATA reagent (Pierce) and 0.36wt. eq. of NaHCO₃ in water at 4° C. for 16 h. The protected thiolmodified siRNAs were precipitated by the addition of 9 volumes ofethanol and incubated at −78° C. for 2 h. The precipitate was isolated,dissolved in 1×siRNA buffer (Dharmacon), and quantitated by measuringthe absorbance at the 260 nm wavelength.

Separately, polymer in 5 mM TAPS, pH 9, was modified by addition of 1.5wt % 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]-toluene(SMPT, Pierce). 1 h after addition of SMPT, the SMPT-polymer was addedto isotonic glucose solution containing 5 mM TAPS pH 9. To this solutionthe SATA-modified siRNA was added. The resulting polynucleotide-polymerconjugating disulfide bond is reversible in the reducing environment ofthe cytoplasm.

The siRNA-polymer conjugate was then masked by adding HEPES free base tothe solution followed by a mixture of CDM-NAG and/or CDM-PEG. Thesolution was then incubated for 1 h at room temperature (RT) beforeinjection.

B. SATA/SPDP linkage. siRNA having a 5′-amino group on the sense strandwas reacted with SATA in the presence of HEPES base pH 7.5. Separately,polymer was reacted with 3-(2-pyridyldithio)propionic acidN-hydroxysuccinimide ester (SPDP) in the presence of HEPES pH 7.5. Themodified siRNA and modified polymer were then combined to allow covalentattachment of the siRNA to the polymer.

C. 5-methyl-2-iminothiolane linkage. siRNA having an strand terminalamino group is reacted with S-acetyl groups to yield siRNA-SAc. Thepolymer is reacted with 5-methyl-2-iminothiolane (M2IT) in the presenceof 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) to yield the polymerhaving an activated disulfide.

The above modified polymer is then reacted with siRNA-SAc to form thesiRNA-polymer conjugate.

C. Maleic anhydride linkage. siRNA having a strand terminal amino groupis reacted with a disubstituted maleic anhydride derivative, such as a2-propionic-3-methylmaleic anhydride, that also contains an additionalamine reactive group, e.g. CDM-thioester, in the presence alkalinebuffer (e.g., HEPES, pH 7.9). To the siRNA-maleic anhydride is added thepoly(vinyl ester). The maleic anhydride then reacts with amines on thepolymer.

Example 9 Reversible Modification (Masking) of Membrane ActivePoly(vinyl ester) Random Copolymers

A. Modification with maleic anhydride-based masking agents. Prior tomodification, 5-7× mg of disubstituted maleic anhydride masking agent(e.g. CDM-NAG) was lyophilized from a 0.1% aqueous solution of glacialacetic acid. To the dried disubstituted maleic anhydride masking agentwas added a solution of × mg polymer in 0.2× mL of isotonic glucose and10× mg of HEPES free base. Following complete dissolution of anhydride,the solution was incubated for at least 30 min at RT prior to animaladministration. Reaction of disubstituted maleic anhydride masking agentwith the polymer yielded:

wherein R is poly(vinyl ester) polymer and R1 comprises a targetingligand or steric stabilizer. The anhydride carboxyl produced in thereaction between the anhydride and the polymer amine exhibits ˜1/20^(th) of the expected charge (Rozema et al. Bioconjugate Chemistry2003). Therefore, the membrane active polymer is effectively neutralizedrather than being converted to a highly negatively charged polyanion.

In some applications, the polymer was modified in a two-step process.First CDM-based masking agents with shielding (PEG) and targeting groupswere mixed in a ratio of 2:1 (wt:wt) shielding to targeting agent. Thepolymer was modified with 2× mg of the CDM masking agents mixture for 30min, followed by attachment of siRNA. The polymer-siRNA conjugate wasthen further modified with 5× mg of the CDM masking agents mixture. Thesolution was then incubated at least 1 h at room temperature (RT) beforeinjection into animals.

B. Modification with protease cleavable masking agents. Activated (aminereactive) carbonates of p-acylamidobenzyl alcohol derivatives arereacted with amino groups of amphipathic membrane active polyamines inH₂O at pH>8 to yield a p-acylamidobenzyl carbamate.

R¹ comprises an targeting group ligand (either protected or unprotected)or a PEG,

R² is an amphipathic membrane active poly(vinyl ester),

AA is a dipeptide (either protected or unprotected), and

Z is an amine-reactive carbonate.

To × mg polymer was added 10-12× mg of HEPES free base in isotonicglucose. To the buffered polymer solution was added 2× to 16× mg 200mg/ml dipeptide masking agent in DMF. In some applications, the polymerwas modified with 2× mg dipeptide masking agent followed by attachmentof siRNA. The polymer-siRNA conjugate was then further modified with 6×to 8× mg dipeptide masking agent. The solution was then incubated atleast 1 h at room temperature (RT) before injection into animals. Insome applications, the polymer was modified with 2× mg PEG dipeptidemasking agent followed by attachment of siRNA. The polymer-siRNAconjugate was then further modified with 6× to 8× mg targeting liganddipeptide masking agent. The solution was then incubated at least 1 h atroom temperature (RT) before injection into animals.

In some applications, the polymer was modified with 2× mg dipeptidemasking agent followed by attachment of siRNA. The polymer-siRNAconjugate was then further modified with 6× to 8× mg CDM-based maskingagent. The solution was then incubated at least 1 h at RT beforeinjection into animals. In some applications, the polymer was modifiedwith 2× mg PEG dipeptide masking agent followed by attachment of siRNA.The polymer-siRNA conjugate was then further modified with 6× to 8× mgtargeting ligand CDM-based masking agent. The solution was thenincubated at least 1 h at room temperature (RT) before injection intoanimals.

Example 10 Conjugate Formation—Masking and Polynucleotide Attachment

A) Polymer was modified with SMPT. After 1 h, 2 wt. equivalents ofCDM-NAG (N-acetylgalactoseamine) and/or CDM-PEG (average 11 units) wasadded to the polymer in the presence of HEPES base. To this solution wasadded SATA-siRNA. After overnight incubation, CDM-NAG and/or CDM-PEG wasadded to the conjugate.

B) Polymer was modified with SMPT. After 1 h, 2 wt equivalents ofPheCit-NAG (N-acetylgalactoseamine) and/or PheCit-PEG (average 11unites) was added to the polymer in the presence of HEPES base. To thissolution was added SATA-siRNA. After overnight incubation, a PheCit-NAGand/or PheCit-PEG was added to the conjugate.

Example 10 siRNAs

The siRNAs had the following sequences:

Factor VII - rodent sense: (SEQ ID 1) 5′GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 3′ antisense: (SEQ ID 2) 5′pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT 3′ or sense (SEQ ID 3) 5′GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT 3′ antisense (SEQ ID 4) 5′GUfAAGACfUfUfGAGAUfGAUfCfCfdTsdT 3′ Factor VII = primate Sense(SEQ ID 5) 5′ uuAGGfuUfgGfuGfaAfuGfgAfgCfuCfaGf(invdT) 3′ Antisense(SEQ ID 6) 5′ pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfcdTsdT 3′ ApoB siRNA: sense(SEQ ID 7) 5′ GGAAUCuuAuAuuuGAUCcAsA 3′ antisense (SEQ ID 8) 5′uuGGAUcAAAuAuAAGAuUCcscsU 3′ siLUC sense (SEQ ID 9)5′-uAuCfuUfaCfgCfuGfaGfuAfcUfuCfgAf(invdT)-3′ antisense (SEQ ID 10)5′-UfcGfaAfgUfaCfuCfaGfcGfuAfaGfdTsdT-3′ lower case =2′-O—CH₃ substitution s = phosphorothioate linkage f after nucleotide =2′-F substitution d before nucleotide = 2′-deoxy

RNA synthesis was performed on solid phase by conventionalphosphoramidite chemistry on an ÄKTA Oligopilot 100 (GE Healthcare,Freiburg, Germany) with controlled pore glass (CPG) as solid support.

Example 11 Synthesis of Amino-Modified RNA

RNA equipped with a C-6-aminolinker at the 5′-end of the sense strandwas produced by standard phosphoramidite chemistry on solid phase at ascale of 1215 μmol using an ÄKTA Oligopilot 100 (GE Healthcare,Freiburg, Germany) and controlled pore glass as solid support (PrimeSynthesis, Aston, Pa., USA). RNA containing 2′-O-methyl nucleotides weregenerated employing the corresponding phosphoramidites, 2′-O-methylphosphoramidites, and TFA-hexylaminolinker amidite (Sigma-Aldrich, SAFC,Hamburg, Germany). Cleavage and deprotection as well as purification wasachieved by methods known in the field (Wincott F., et al, NAR 1995, 23,14, 2677-84).

Example 12 In vivo Delivery of RNAi Polynucleotides using Poly(vinylester) Delivery Polymers

RNAi polynucleotide conjugates and masked poly(vinyl ester) polymerswere synthesized as described above. Six to eight week old mice (strainC57BL/6 or ICR, ˜18-20 g each) were obtained from Harlan Sprague Dawley(Indianapolis, Ind.). Mice were housed at least two days prior toinjection. Feeding was performed ad libitum with Harlan Teklad RodentDiet (Harlan, Madison Wis.). Mice were injected by infusion into thetail vein with 0.4 mL solution of delivery polymer-siRNA conjugates intothe tail vein unless stated otherwise. The composition was soluble andnonaggregating in physiological conditions. Injection into othervessels, e.g. retro-orbital injection, are predicted to be equallyeffective.

Wistar Han rats, 175-200 g were obtained from Charles River (Wilmington,Mass.). Rats were housed at least one (1) week prior to injection.Injection volume for rats was typically 1 ml.

The indicated amount of polymer-siRNA conjugate was administered toCynomolgus macaque (Macaca fascicularis) primates (male, 3.0 to 8.0 kg)via injection into the saphenous vein using a 22 to 25 gauge intravenouscatheter. As a control, another set of primates were injected withisotonic glucose. Blood tests for blood urea nitrogen (BUN), alaninetransaminase (ALT), aspartate aminotransferase (AST), and creatininewere performed on a Cobas Integra 400 (Roche Diagnostics) according tothe manufacturer's recommendations.

Mice, rats, and primates were fasted for 4 h, 16 h, or overnight, beforeinjection. Primates were fasted overnight before blood collection ortissue harvest. Blood samples were collected by submandibular bleedingfor mice, from jugular vein for rats, and from femoral vein forprimates. For mice and rats, samples were taken 2 days after polymerinjection, unless indicated otherwise. For primates, blood samples arecollected on day 2 (24 h after injection) and day 4 (72 h afterinjection). Further, for primates, blood sample collections were carriedout up to day 81. Serum for use in Western assays was collected andadded to an equal volume of Complete Protease Inhibitor Cocktailcontaining EDTA (Roche, Indianapolis Ind.) and stored at −20° C. TotalRNA was isolated from liver immediately after harvest using TRI-REAGENT®according to the manufacturer's protocol (Molecular Research Center,Cincinnati Ohio).

Serum ApoB levels determination. Serum ApoB protein levels weredetermined by standard sandwich ELISA methods. Briefly, a polyclonalgoat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB antibody(Biodesign International) were used as capture and detection antibodiesrespectively. An HRP-conjugated goat anti-rabbit IgG antibody (Sigma)was applied afterwards to bind the ApoB/antibody complex. Absorbance oftetramethyl-benzidine (TMB, Sigma) colorimetric development was thenmeasured by a Tecan Safire2 (Austria, Europe) microplate reader at 450nm.

Plasma Factor VII (F7) activity measurements. Plasma samples fromanimals were prepared by collecting blood (9 volumes) (by submandibularbleeding for mice or from jugular vein for rats) into microcentrifugetubes containing 0.109 mol/L sodium citrate anticoagulant (1 volume)following standard procedures. F7 activity in plasma is measured with achromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason,Ohio) following manufacturer's recommendations. Absorbance ofcolorimetric development was measured using a Tecan Safire2 microplatereader at 405 nm.

Example 13 Factor VII Knockdown in Mouse, Rat, and Non-human PrimateFollowing Factor VII siRNA Delivery by P(BAVVE-co-VBu) Polymer

P(BAVVE-co-VBu) polymer (DAN-41947-106-1 or DAN-41947-129-1) wasreversibly modified with 2.3 wt equivalents of CDM-NAG and 4.7 wtequivalents CDM-PEG and conjugated to Factor VII siRNA (duplex of SEQ ID3 and 4) as described above. Effect on Factor VII levels was determinedas described above. Effective knockdown of Factor VII activity wasobserved using about 0.5 mg/kg to nearly 16 mg/kg P(BAVVE-co-VBu)polymer without causing a more than 3-fold increase in blood ureanitrogen (BUN), alanine transaminase (ALT), aspartate aminotransferase(AST), or creatinine levels.

TABLE 4 Inhibition of Factor VII activity in normal liver cells inanimal treated with CDM-NAG/CDM-PEG masked P(BAVVE-co-VBu) polymerconjugated to Factor VII-siRNA. % target gene knockdown siRNA polymernon-human polymer mg/kg mg/kg mouse rat primate DAN-41947-106-1 2 7.5 8115 100 0.2 0.5 68 1 87 5 81 8 86 10 91 12 83 16 74 0.8 12 97DAN-41947-129-1 0.125 0.5 80 0.25 1 97 2 7.5 70 0.2 0.5 80 0.2 12 85

Example 14 Target Gene Knockdown in Mouse Following siRNA Delivery usingPoly(vinyl ester) Delivery Polymers with Varying Monomer RatioComposition

P(BAVVE-co-VBu) or P(BAPVE-co-VBu) polymers with varying aminemonomer:hydrophobic monomer compositions were reversibly modified with2.3 wt equivalents of CDM-NAG and 4.7 wt equivalents CDM-PEG andconjugated to Factor VII siRNA (duplex of SEQ ID 3 and 4 above) asdescribed above. 7.5 mg/kg polymer and 2 mg/kg siRNA were injected intoeach mouse. Effect on Factor VII levels were determined as describedabove. The results demonstrate that effective knockdown was observedwith polymers having 56-80% amine content. However, polymers having56-60% amine content were the most efficient.

TABLE 5 siRNA delivery to normal liver cells in mice treated with maskedP(BAVVE-co-VBu) or P(BAPVE-co-VBu) delivery polymers as determined bytarget gene knockdown. % target hydro- gene amine phobic knock- polymermonomer monomer ratio M_(n, th) down DAN-42435-15-A-1 valeric butyryl56:44 200 KDa 90% DAN-41947-106-1 valeric butyryl 60:40 200 KDa 81%DAN-42435-16-A-1 propionyl butyryl 70:30 200 KDa 70% DAN-42435-14-B-1valeric butyryl 75:25 200 KDa 60% DAN-42435-14-A-1 valeric butyryl 80:20200 KDa 40%

Example 15 Target Gene Knockdown in Mouse Following siRNA Delivery UsingPoly(vinyl ester) Delivery Polymers Formed with Different Amine Monomers

P(BAPVE-co-VBu), P(BABVE-co-VBu), and P(BAVVE-co-VBu) polymers werereversibly modified with 2.3 wt equivalents of CDM-NAG and 4.7 wtequivalents CDM-PEG and conjugated to Factor VII siRNA (duplex of SEQ ID3 and 4 above) as described above. 7.5 mg/kg polymer and 2 mg/kg siRNAwere injected into each mouse. Effect on Factor VII levels wasdetermined as described above. The results demonstrate that effectiveknockdown was observed with each of the amine monomers tested.

TABLE 6 siRNA delivery to normal liver cells in mice treated with maskedP(BAPVE-co-VBu), P(BABVE-co-VBu), and P(BAVVE-co-VBu) delivery polymersas determined by target gene knockdown. hydro- mono- % target phobic mergene amine mono- feed knock- polymer monomer mer ratio M_(n, th) downDAN-42435-16-B-1 propionyl butyryl 60:40 200 KDa 90% DAN-41947-96-1butyryl butyryl 60:40 200 kDa 84% DAN-41947-106-1 valeric butyryl 60:40200 KDa 81%

Example 16 Target Gene Knockdown in Mouse Following siRNA Delivery usingPoly(vinyl ester) Delivery Polymers with Different Hydrophobic Monomers

P(BAVVE-co) polymers with different hydrophobic monomers were reversiblymodified with 2.3 wt equivalents of CDM-NAG and 4.7 wt equivalentsCDM-PEG and conjugated to Factor VII siRNA (duplex of SEQ ID 3 and 4above) as described above. 7.5 mg/kg polymer and 2 mg/kg siRNA wereinjected into each mouse. Effect on Factor VII levels was determined asdescribed above. The results demonstrate that effective knockdown wasobserved with butyryl and valeryl hydrophobic monomers. Polymers havingbutyryl hydrophobic monomers were more efficient.

TABLE 7 siRNA delivery to normal liver cells in mice treated with maskedP(BAVVE-co-hydrophobic vinyl ester) delivery polymers as determined bytarget gene knockdown. amine hydrophobic % target gene polymer monomermonomer knockdown DAN-41947-110-1 valeryl propionyl (C3) 0%DAN-41947-106-1 valeryl butyryl (C4) 81% DAN-41947-123-B-1 valerylvaleryl (C5) 30% DAN-42435-82-A-1 valeryl valeryl (C5) 50%DAN-42435-78-A-1 valeryl hexanyl (C6) 0% DAN-42435-80-A-1 valeryloctanyl (C8) 0%

Example 17 Target Gene Knockdown in Mouse Following siRNA Delivery UsingPoly(vinyl ester) Delivery Polymers with Varying Molecular Weights

P(BAVVE-co-VBu) polymers with varying molecular weights were reversiblymodified with 2.3 wt equivalents of CDM-NAG and 4.7 wt equivalentsCDM-PEG and conjugated to Factor VII siRNA (duplex of SEQ ID 3 and 4above) as described above. 7.5 mg/kg polymer and 2 mg/kg siRNA wereinjected into each mouse. Effect on Factor VII levels was determined asdescribed above. The results demonstrate that effective knockdown wasobserved with P(BAVVE-co-VBu) polymers having 23k-200k M_(n, th).Polymers having M_(n, th) greater than 67 kDa (70 kDa Mw) were moreefficient. Polymers with 100-150 kDa (67-86 kDa Mw) were most preferred.

TABLE 8 siRNA delivery to normal liver cells in mice treated with maskedP(BAVVE-co-VBu) delivery polymers with varying molecular weight asdetermined by target gene knockdown. % target gene polymer M_(n, th) Mwknockdown DAN-41947-47-A-1  30 kDa 23150 20% DAN-41947-47-B-1  50 kDa42370 50% DAN-41947-47-C-1  75 kDa 67960 80% DAN-41947-47-D-1 100 kDa76600 90% DAN-41947-47-E-1 150 kDa 86100 90% DAN-41947-106-1 200 kDa 81%

Example 18 Amphipathic Cationic Poly(vinyl ester) Random Copolymers areEffective In Vitro Transfection Reagents

The indicated copolymer (500 mg) was dissolved in a solution of 2 N HClin acetic acid (5 mL) and stirred for 1 h. The solution was diluted withwater (30 mL) and dialyzed against an aqueous NaCl solution and thendeionized water over two days. The solution was then lyophilized andre-dissolved in H₂O to make up 20 mg/mL solutions. Hep3B-SEAP(hepatocellular carcinoma), MCF7 (breast cancer), HT29 (colon cancer),HepG2-SEAP (hepatocellular carcinoma), or A375 (melanoma) cells asindicated were plated in 96-well culture plates at a density of 10,000cells/well. Cells were transfected with either 1.5 μg/mL or 3 μg/mL ofcopolymer and 500 ng/mL of Ahal siRNA prepared in OPTI-MEM reduced-serummedium (Gibco). 24 h post-transfection, the cells were lysed andprocessed for quantitative real-time PCR (qRT-PCR) using the TaqMan GeneExpression Cells-to-CT Kit (Life Technologies). Biplex qRT-PCR wasperformed using TaqMan assays for human Aha1 (product # Hs00201602_ml)and human CycA (product # 4326316E) on a StepOne Real-Time PCR System(Life Technologies). Analysis was performed using the ΔΔCT method ofrelative gene expression quantitation.

TABLE 9 Poly(vinyl ester) polymers used for in vitro transfection.polymer short name long name % Amine MW^(a) BAPVE-VBu3-tert-Butoxycarbonylamino- 50 23.9 propionic vinyl ester + vinylbutyrate 50 57.5 62 69.7 63 27.0 63 67.6 68 63.4 71 23.3 71 59.0BAPVE-VV 3-tert-Butoxycarbonylamino- 62 17.5 propionic vinyl ester +vinyl valerate 67 15.4 78 17.2 92 50.4 ^(a)kDa

TABLE 10 Knockdown of Aha1 in vitro following Aha1 siRNA by poly(vinylester) polymers. Polymers are listed in same order as in Table 9. % Aha1Knockdown Hep3B- HepG2- cell type SEAP MCF7 HT29 SEAP A375 polymerdose^(a) 3.0 1.5 3.0 1.5 3.0 1.5 3.0 1.5 3.0 1.5 BAPVE-VBu 69 86 33 4917 9 55 53 68 68 83 90 37 53 14 9 70 58 80 80 52 75 34 23 17 6 34 36 5166 61 80 33 25 5 −7 34 36 56 54 72 74 41 27 9 −13 50 49 62 57 55 70 3619 14 12 39 29 58 40 33 38 36 13 1 −8 4 7 33 23 43 31 39 21 8 −16 13 1560 28 BAPVE-VV 81 90 51 46 17 13 57 68 67 80 41 46 23 15 16 10 27 23 7169 36 55 22 26 16 12 26 21 71 64 48 76 34 35 29 19 45 40 82 79 ^(a)μg/mL

Example 19 siRNA Delivery In Vivo upon Subcutaneous Injection usingNAG/PEG-AA-p-nitrophenyl-carbamate Poly(vinyl ester) DPCs

Polyvinylester DAN-41947-129-1 in 100 mM pH 7.5 HEPES buffer wasmodified 0.5 wt % with the activated disulfide reagentsuccinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene(SMPT) from Pierce. The thiol-reactive polymer was diluted to 5 mg/mL in60 mg/mL HEPES base. To this solution was added 10 mg/mL PEG(12unit)-Phe-Cit-p-nitrophenyl-carbonate masking reagent. After 1 hour,acetate-protected thiol Factor VII siRNA was added to polymer solutionat a polymer to siRNA ratio range of 5-10 to 1. After incubationovernight, NAG-Ala-Cit-p-nitrophenyl-carbonate masking reagent (FIG. 2)was added to 30 mg/mL. After incubation for at least 60 minutes, but nolonger than 4 hours, the DPC was injected subcutaneously in the areabehind the neck of 20 g ICR mice. At various times after injection, asample of serum was harvested and the levels of fVII were measured. As acontrol similarly prepared DAN-41947-129-1-siRNA conjugate was injectedintravascularly.

TABLE 11 Knockdown of Factor VII in vivo in mice treated withPEG₁₂-Phe-Cit/ NAG-Ala-Cit-p-nitrophenyl-carbamate DPCs Days PolymersiRNA Post- dose dose % fVII Masking Agent injection (mg/kg)^(a)(mg/kg)^(a) activity^(b) 2 wt equivalents 12 unit 3 25 2.5 3PEG12-PheCit followed by 5 25 2.5 2 6 wt NAG-PheCit 7 25 2.5 2 2 wtequivalents 12 unit 5 25 2.5 2 PEG24-PheCit followed by 6 wt NAG-AlaCitI.V. injection control 2 wt equivalents 12 unit 2 10 0.5 27 PEG14-PheCitfollowed by 6 wt NAG-AlaCit ^(a)mg polymer or siRNA per kg animal weight^(b)relative to naive control

Example 20 Inhibition of Endogenous Gene Expression in In Vivo FollowingCo-administration of Cholesterol-siRNA and Masked Amphipathic Poly(vinylester) Random Copolymers

The poly(vinyl ester) polymer DAN-41947-129-1 were masked bydisubstituted maleic anhydride masking agents or didpetide maskingagents as described above. The masked polymers were then co-injectedwith cholesterol-siRNA (ApoB or Factor VII) into mice and the effect ontarget gene expression was determined.

TABLE 12 DAN-41947-129-1 (15 mg/kg) masked with CDM-PEG and CDM-NAG orPEG(12)-PheCit and NAG-AlaCit was co-injected with cholesterol conjugatesiRNA mice. 48 h after injection, ApoB knockdown was measured. siRNAinjection % gene masking agent gene μg volume activity^(a) 4.7 × CDM-PEG2.3 × CDM-NAG ApoB 40 200 μL 1 2 × PEG(12)- 6 × NAG-AlaCit fVII 100 300μL 3 PheCit ^(a)relative to isotonic glucose injection control

The invention claimed is:
 1. A membrane active poly(vinyl ester) randomcopolymer having the structure represented by:

wherein: N is —NH₂ or —N—CO—O—C—(CH₃)₃ N′ is —NR⁵H, —NR⁵R⁶, —NR⁵R⁶R⁷,nitrogen heterocycle, aldimine, hydrazide, hydrazone, or imidazole,wherein R⁵, R⁶, and R⁷ are independently selected from —CH₃ and—CH₂—CH₃, Y and Y′ are independently —(CH₂)_(a)— or—(CH₂—CH₂—0)_(b)—(CH₂)_(c)— wherein a , b, and c are independently 1, 2,3, 4, 5, or 6, R is a hydrophobic group having 1-7 carbon atoms or analkoxy ethyl group, R′ is a hydrophobic group having 12-20 carbon atoms,R1, R2, R3, and R4 are independently selected from hydrogen (—H) andmethyl (—CH₃), m and p are independently integers greater than zero (0),n and q are independently integers greater than or equal to zero (0),the ratio (m+n)/(p+q) is 1-9, and the poly(vinyl ester) random copolymeris is capable of causing red blood cell lysis.
 2. The poly(vinyl ester)random copolymer of claim 1 wherein the ratio (m+n)/(p+q) is 1.5-4. 3.The poly(vinyl ester) random copolymer of claim 1 wherein thepolydispersity of the polymer is less than 1.5.
 4. The poly(vinyl ester)random copolymer of claim 1 wherein Y is —(CH₂)_(a)— wherein a is 2, 3,or
 4. 5. The poly(vinyl ester) random copolymer of claim 1 wherein Y is—CH₂—CH₂—O—CH₂—CH₂—.
 6. The poly(vinyl ester) random copolymer of claim1 wherein R is —(CH₂)_(k)—CH₃ wherein k is 1, 2, 3, 4, or
 6. 7. Thepoly(vinyl ester) random copolymer of claim 1 wherein n is zero.
 8. Thepoly(vinyl ester) random copolymer of claim 7 wherein q is zero.
 9. Thepoly(vinyl ester) random copolymer of claim 1 wherein R1, R2, R3 and R4are each hydrogen.
 10. The poly(vinyl ester) random copolymer of claim 1wherein: Y is —(CH₂)₄— R is —(CH₂)₂—CH₃ R1 and R3 are each hydrogen, andn and p are each zero.
 11. The poly(vinyl ester) random copolymer ofclaim 10 wherein ratio m/p is 1.3-2.3.
 12. The poly(vinyl ester) randomcopolymer of claim 1 wherein: Y is —(CH₂)₂— R is —(CH₂)₂—CH₃ R1 and R3are each hydrogen, and n and p are each zero.
 13. The poly(vinyl ester)random copolymer of claim 1 wherein: Y is —(CH₂)₂— R is —(CH₂)₃—CH₃ R1and R3 are each hydrogen, and n and p are each zero.
 14. The poly(vinylester) random copolymer of claim 1 wherein the polymer is conjugated toan RNA interference polynucleotide.
 15. The poly(vinyl ester) randomcopolymer of claim 1 wherein greater than 50% of N are reversiblymodified by reaction with disubstituted maleic anhydride masking agents,dipeptide-amidobenzyl-carbonate masking agents, or a combination ofdisubstituted maleic anhydride masking agents anddipeptide-amidobenzyl-carbonate masking agents.
 16. A composition forinhibiting gene expression in vivo comprising the poly(vinyl ester)random copolymer of claim 1 and an RNA interference polynucleotide in apharmaceutically acceptable carrier.
 17. The composition of claim 16wherein the RNA interference polynucleotide is covalently linked to thepoly(vinyl ester) random copolymer.
 18. The composition of claim 16wherein the RNA interference polynucleotide is conjugated to ahydrophobic group containing at least 20 carbon atoms.
 19. A membraneactive poly(vinyl ester) random copolymer having the structurerepresented by:

wherein: N is a primary amine having the form —NH₂, Y is —(CH₂)_(a)— or—(CH₂—CH₂—O)_(b)—(CH₂)_(c)— wherein a , b, and c are independently 1, 2,3, 4, 5, or 6, R is a hydrophobic group having 1-7 carbon atoms, R1 andR2 are independently selected from hydrogen (—H) and methyl (—CH₃), m isan integer greater than zero (0), p is an integer greater than zero (0),the ratio m/p is 1-9, and the poly(vinyl ester) random copolymer is iscapable of causing red blood cell lysis.
 20. The poly(vinyl ester)random copolymer of claim 19 wherein the ratio m/p is 1.5-4.